Basic Study
Copyright ©The Author(s) 2016. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Hepatol. Oct 18, 2016; 8(29): 1222-1233
Published online Oct 18, 2016. doi: 10.4254/wjh.v8.i29.1222
Lycopene modulates cellular proliferation, glycolysis and hepatic ultrastructure during hepatocellular carcinoma
Prachi Gupta, Nisha Bhatia, Mohinder Pal Bansal, Ashwani Koul
Prachi Gupta, Nisha Bhatia, Mohinder Pal Bansal, Ashwani Koul, Department of Biophysics, Basic Medical Sciences Block II, Panjab University, Chandigarh 160014, India
Author contributions: All authors contributed to the manuscript.
Supported by University Grant Commission, New Delhi, No. 2060930310.
Institutional review board statement: The study was reviewed and approved by the Institutional Animal Ethics Committee (IAEC) of Panjab University, Chandigarh (India) and conducted according to the Indian National Science Academy guidelines for the use and care of experimental animals (IAEC/284-295 at Sr. No. 48).
Institutional animal care and use committee statement: All procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of the Panjab University, Chandigarh, India [IACUC protocol number: (IAEC/284-295 at Sr. No. 48)].
Conflict-of-interest statement: The authors declare that there are no conflicts of interest.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Ashwani Koul, Professor, Department of Biophysics, Basic Medical Sciences Block II, Panjab University, South Campus, Sec-25, Chandigarh 160014, India. drashwanikoul@yahoo.co.in
Telephone: +91-172-2534119
Received: May 4, 2016
Peer-review started: May 5, 2016
First decision: June 6, 2016
Revised: June 20, 2016
Accepted: July 20, 2016
Article in press: July 22, 2016
Published online: October 18, 2016

Abstract
AIM

To investigate the effect of lycopene extracted from tomatoes (LycT) on ultrastructure, glycolytic enzymes, cell proliferation markers and hypoxia during N-Nitrosodiethylamine (NDEA)-induced hepatocarcinogenesis.

METHODS

Female BALB/c mice were randomly divided into four groups: The Control, NDEA (200 mg NDEA/kg b.w. given i.p.), LycT (5 mg/kg b.w. given orally on alternate days) and LycT + NDEA group. The mRNA and protein expression of various cell proliferation markers (PCNA, Cyclin D1, and p21) were assessed by reverse transcription-polymerase chain reaction and enzyme linked immunosorbent assay, respectively. The ultrastructure of hepatic tissue was analyzed using scanning and transmission electron microscopy. The enzymatic activity of glycolytic enzymes was estimated using standardized protocols, while glucose-6-phosphate dehydrogenase activity level was estimated using a kit obtained from Reckon Diagnostic P. Ltd. (India).

RESULTS

Uncontrolled proliferation in the liver of NDEA (P ≤ 0.001) mice was evident from the high expression of cell-proliferation associated genes (PCNA, Cyclin D1, and p21) when compared to control and LycT mice. In addition, enhanced activities of hexokinase, phosphoglucoisomerase, aldolase, glucose-6-phosphate dehydrogenase and hypoxia-inducible factor-1α were observed in NDEA mice as compared to control (P ≤ 0.001) and LycT (P ≤ 0.001) mice. The alterations in hepatic ultrastructure observed in the NDEA group correlated with the changes in the above parameters. LycT pre-treatment in NDEA-challenged mice ameliorated the investigated pathways disrupted by NDEA treatment. Moreover, hepatic electron micrographs from the LycT + NDEA group showed increased macrophages, apoptotic bodies and well-differentiated hepatocellular carcinoma (HCC) in comparison to undifferentiated HCC as observed in the NDEA treated group.

CONCLUSION

This study demonstrates that dietary supplementation with LycT has a multidimensional role in preventing HCC development.

Key Words: Hepatocellular carcinoma, Ultrastructure, Hypoxia, Cell proliferation, Lycopene, Glycolysis

Core tip: The present study was designed to evaluate the chemopreventive role of lycopene extracted from tomatoes (LycT) against N-Nitrosodiethylamine-induced hepatocellular carcinoma (HCC). The findings suggested the mechanism underlying LycT-mediated chemoprevention of HCC.


Citation: Gupta P, Bhatia N, Bansal MP, Koul A. Lycopene modulates cellular proliferation, glycolysis and hepatic ultrastructure during hepatocellular carcinoma. World J Hepatol 2016; 8(29): 1222-1233
INTRODUCTION

The continuous rising trend in cancer worldwide necessitates potential action against this deadly disease. Cancer is a complex disease and requires attention in multiple directions to prevent its development. Nearly two-thirds of all cancer cases are linked to inadequate components in the diet, environmental exposure to pollutants and occupational exposure to toxic materials[1,2]. Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and is the second most common cause of cancer-related death[3]. N-Nitrosodiethylamine (NDEA) is a known potent environmental hepatic carcinogen and has been used as an initiator in several hepatic cancer models[4,5]. Besides direct exogenous exposure, humans are also exposed to endogenously produced nitrosamines[6]. NDEA is metabolized in the liver to its active ethyl radical metabolite and various other reactive metabolites, which are highly reactive towards DNA, proteins and lipids, thus exhibiting sequential cellular and molecular alterations leading to its hepatocarcinogenic effect[4,7-9].

Natural and experimental chemical hepatocarcinogenesis is accompanied by altered cellular redox status, altered cytochemical pathways, altered molecular phenomena, chromosomal instability and altered physiological environment in cells. Of the various carcinogenic insults, uncontrolled proliferation, dysregulated carbohydrate metabolism and hypoxia play very crucial roles in HCC development. Aerobic glycolysis and diversion to a biosynthetic pathway, i.e., the pentose phosphate pathway are the metabolic hallmarks of carcinogenesis[10]. In recent years, interest in these pathways has been renewed in the tumor microenvironment which has a profound effect on core tumor metabolism. Hypoxia inducible factor-1α (HIF-1α) is involved in many compensatory pathways such as angiogenesis, glucose metabolism, survival and tumor development[11]. Although there have been advances in therapeutic approaches a complete cure is still unavailable. Dietary multi-targeted agents have attracted the attention of cancer biologists as these agents may provide a solution to this complex problem by targeting multiple targets simultaneously[12]. A large body of evidence has revealed an association between phytochemicals and a reduced risk of developing chronic diseases[13,14].

Lycopene, a polyunsaturated hydrocarbon imparting red colour to various fruits is a nutritionally important carotenoid exhibiting beneficial health effects by virtue of its antioxidant activity with minimal side effects[15]. A large number of studies have shown an association between lycopene and a reduced risk of developing chronic diseases such as cancer, diabetes, cardiovascular disorders and degenerative diseases[15-18]. In addition to its antioxidant property, lycopene is known to modulate other non-oxidative pathways such as regulation of gap junction communication, the hormonal system, the immune system and the metabolic pathways of xenobiotics[16,19]. Phytochemicals also tend to be more effective for long-standing health problems that do not respond well to synthetic medicines[20].

In our previous studies we developed a standardized detailed protocol for lycopene extracted from tomatoes (LycT), its characterization and its beneficial effect in inhibiting NDEA-induced HCC development in terms of histopathological observations, tumor statistics, apoptosis, antioxidative capacity and toxicity[21-23]. However, further studies are warranted to determine the modulating effect of lycopene on dysregulated glucose metabolism and hypoxia, as these processes play critical roles in cancer. Thus, the present study was designed to explore the influence of LycT on various glycolytic and non-glycolytic enzymes, the expression of HIF-1α and potent cell proliferation-associated genes, while preventing NDEA-induced HCC. Moreover, an attempt was made to demonstrate the impact of an imbalance between energy production and metabolic demands on the gross morphology and ultrastructure of hepatocytes in HCC.

MATERIALS AND METHODS
Chemicals

Azino-bis(ethylbenzthiazoline sulfonic acid) (ABTS), diaminobenzidine, ethidium bromide, TRI-reagent and NDEA were obtained from Sigma Chemicals (St. Louis, MO, United States). Primary and secondary antibodies were obtained from Santa Cruz Biotechnology, CA, United States. Invitrogen superscript (III) one step reverse transcription-polymerase chain reaction (RT-PCR) was purchased and used for RT-PCR analysis. Other chemicals were purchased from local reputable companies including Sisco Research Laboratory (P) Ltd. Detailed information regarding extraction and characterization of LycT has been reported previously (Gupta et al[21]).

Animal model and experimental conditions

The animal protocol was designed to minimize pain or discomfort to the animals. All animals were acclimatized to laboratory conditions, i.e., temperature of 21 °C ± 1 °C and humidity of 50%-60% for one week prior to the various treatments. All the mice were provided with drinking water and a standard animal pellet diet ad libitum. Female BALB/c mice (25-30 g) were randomly divided into four groups (n = 7 per group). Animals in Group I (Control) received 0.1 mL olive oil (vehicle) orally throughout the experiment. Group II (NDEA) animals received a cumulative dose of 200 mg NDEA/kg body weight (b.w.) given intraperitoneally in 8 wk as described previously[5]. Group III (LycT) mice received LycT orally at a dose of 5 mg/kg b.w. thrice a week for 24 wk. Group IV (LycT + NDEA) animals received NDEA in the same manner as Group III and were also given LycT at a dose of 5 mg/kg b.w. thrice a week for 24 wk. LycT administration was commenced two weeks prior to NDEA treatment. Animals were sacrificed after the 24th week to evaluate the modulatory effect of LycT in NDEA-induced hepatocarcinogenesis. The experimental study was reviewed and approved by the Institutional Animal Ethics Committee (IAEC) of Panjab University, Chandigarh (India) and conducted according to the Indian National Science Academy guidelines for the use and care of experimental animals (IAEC/284-295 at Sr. No. 48).

Scanning and transmission electron microscopy

After 24 wk, liver tissues from animals in the different groups were immediately fixed in 2% para-formaldehyde and 2.5% glutaraldehyde prepared in 100 mmol/L phosphate buffer (pH 7.4) for 6 h at 4 °C. Critical point drying, trimming of the tissue and gold coating were carried out and the tissues were viewed under a LEO 435 VP scanning electron microscope. Secondary fixation, treatment with a mixture of propylene oxide and epoxy resin (1:1), embedding in freshly prepared epoxy resin and ultrathin sections mounted on colloid on-carbon coated grids were carried out and examined with a Philips CM-10 transmission electron microscope.

Estimation of the activities of glycolytic enzymes, glucose-6-phosphate dehydrogenase and glycogen content

The specific activity of hexokinase, phosphoglucoisomerase (PGI) and aldolase was estimated according to the reported standard protocols[24-26]. Glucose-6-phosphate dehydrogenase (G6PD) activity level was estimated in a tissue homogenate using an ENZOPAK G6PD kit obtained from Reckon Diagnostic P. Ltd. (India). The activity of G6PD in the samples was further calculated using 6.22 mmol/L per centimeter as the extinction coefficient of NADPH at 340 nm. The glycogen content in liver was estimated using the protocol described by Seifter et al[27]. The amount of glycogen in the aliquot was determined using a glucose standard.

mRNA expression analysis

Total RNA isolation from liver tissue was carried out using TRI-reagent. For RT-PCR analysis, primers for HIF-1α, PCNA, p21 and Cyclin D1 were searched from the database “Gene Runner” and were synthesized by Sigma-Aldrich (United States). The lengths of the primers chosen were approximately 20bp (Table 1). RT-PCR was performed according to the described protocol of the Superscript (III) one step RT-PCR kit. The DNA bands were visualized in agarose gel using an ultraviolet transilluminator and photographed on Gel Doc. Densitometric analysis of the bands was performed using Image J software (National Institute of Health, United States).

Table 1 List of primer pairs used.
GeneStrandPrimer
HIF-1αSense5’-GGT/CAG/ATG/ATC/AGA/GTC/C-3’
Antisense5’-TGC/TTG/GTG/CTG/ATT/TGTG/A-3’
PCNASense5’-GAT/GTG/GAG/CAA/CTT/GGA/AT-3’
Antisense5’-AGC/TCT/CCA/ACT/TGC/AGA/AAA-3’
p21Sense5’-CCG/TGG/ACA/GTG/AGC/AGT/TG -3’
Antisense5’-TGG/GCA/CTT/CAG/GGT/TTT/CT-3’
Cyclin D1Sense5’-CAC/AAC/GCA/CTT/TCT/TTC/CA-3’
Antisense5’-GAC/CAG/CCT/CTT/CCT/CCA/C-3’
β-actinSense5’-ATC/CGT/AAA/GAC/CTC/TAT/GC-3’
Antisense5’-AAC/GCA/GCT/CAG/TAA/CAG/TC-3’
Quantitation of protein expression

Sample preparation: The animals were fasted overnight before liver dissection. Mice were euthanized by cervical dislocation under light ether anesthesia. Liver perfusion was carried out with 0.9% NaCl and the liver was carefully removed and placed in a Petri plate containing ice-cold saline. The tissue was homogenized in ice-cold 100 mmol/L potassium phosphate buffer (pH 7.4) containing 150 mmol/L KCl in an ice-chamber to obtain 25% homogenate (w/v) using a mechanically driven Teflon fitted Potter Elveihem homogenizer. The homogenate (25%) was then subjected to centrifugation at 10000 rpm for 30 min at 4 °C for preparation of the post-mitochondrial fraction.

ELISA: Post mitochondrial fractions obtained from the hepatic tissue of different groups were quantitated for protein concentration by the method of Lowry et al[28]. Two point five microgram protein was loaded onto an ELISA strip containing carbonate buffer. Further, protein expression of HIF-1α, PCNA, p21 and Cyclin D1 were analyzed according to the standard protocol of ELISA using specific primary antibodies and enzyme conjugated secondary antibodies. ABTS in citrate buffer was added along with hydrogen peroxide for color generation. The color thus obtained was quantified at 405 nm.

Statistical analysis

The statistical methods used in this study were reviewed by Dr. Neha Arora Chugh, Department of Biophysics, Panjab University, Chandigarh. Data were expressed as mean ± SD. The results were subjected to analysis of variance (one-way ANOVA) followed by the post hoc test for statistical significance using SPSS (version 14.0) software. P ≤ 0.05 was considered statistically significant.

RESULTS

Scanning electron microscopy (SEM) of the control and LycT groups revealed normal hepatic surface morphology with polyhedral hepatocytes radially arranged around central veins in cords separated by sinusoids (Figure 1A-C). Bile canaliculi were observed on the apical surface of hepatocytes. A few red blood cells were also visible in the sinusoids. However, serious and irreversible alterations in liver architecture were observed in the NDEA and LycT + NDEA groups. Smoothing or rounding of the hepatocytes with hyperplastic tumor along with clumps of hepatocytes with intercellular surfaces covered with numerous microvillus projections were visible (Figure 1D-E). The discernible nodules were of irregular shape and size. Necrotic tumor nodules and uncontrolled cell density revealed the presence of undifferentiated HCC in the NDEA group. In contrast, the surface morphology of liver tissue from the LycT + NDEA group revealed well differentiated HCC characterized by stromal invasion and a trabecular pattern of two to three cells thick plates of hepatocytes (Figure 1H). High cell density with pleomorphism of tumor cells was also evident (Figure 1F). Apoptotic bodies were observed indicating a high rate of apoptosis (Figure 1G). Thus, LycT pre-treatment of NDEA-challenged mice significantly reduced the severity caused by NDEA. Table 2 shows the results of a quantitative comparison of hepatic tissues from the NDEA and LycT + NDEA groups using SEM.

Figure 1
Figure 1 Scanning electron micrographs of liver tissue at × 500 and × 1500. A and B: Control group, illustrating the central vein (CV), hexagonal hepatocytes (encircled) and biconcave RBCs in sinusoids (arrowed); C: LycT group illustrating hexagonal hepatocytes with bile canaliculi (BC) on theits surface (arrowed); D and E: NDEA group illustrating tumor nodules (encircled) with abnormal cell proliferation and disturbed ultrastructure; F-H: LycT + NDEA group respectively illustrating high cell density, CV, apoptotic bodies (arrowed) along with various alterations and two-three cell plate thickening indicating well-differentiated hepatocellular carcinoma, respectively. LycT: Lycopene extracted from tomatoes; NDEA: N-Nitrosodiethylamine; RBCs: Red blood cells.
Table 2 Comparative analysis of the N-Nitrosodiethylamine and lycopene extracted from tomatoes + N-Nitrosodiethylamine group by scanning electron microscopy.
Groups/parametersNDEALycT + NDEA
Cell density++++
Rounding of hepatocytes++++
Trabecular structures-+++
Necrotic tumor nodules++++
Apoptotic bodies-+++
Type of HCCUndifferentiatedWell-differentiated

Transmission electron microscopy (TEM) of the control and LycT groups revealed normal hepatic ultrastructural architecture (Figure 2A-C). At low magnification, hexagonal hepatocytes radially arranged around blood vessels with an intact cell membrane, clear and granulated cytoplasm comprising oval-shaped mitochondria, rough endoplasmic reticulum (RER) and smooth endoplasmic reticulum were observed. A smooth, rounded and prominent nucleus with intact double layered nuclear membrane, a darkly stained single and prominent nucleolus along with uniformly distributed chromatin in the nucleoplasm were also visible. Irregular non-membrane bound, faintly stained granules of hepatocellular glycogen were also observed. The presence of lipid granules as darkly stained spots and a few bi-nucleated hepatocytes were found in the LycT group in addition to the above features. Several irreversible alterations in the nucleus of liver cells from the NDEA group were observed (Figure 2D-E). A prominent large and irregular nucleus with interrupted nuclear membrane, multiple prominent nuclei and pseudo-inclusions were observed in liver sections from the NDEA group with an increased nuclear/cytoplasmic ratio. Karyotin (reticular material) deposition along the nuclear membrane was the striking difference when compared with normal nuclei. Loss of organization of cytoplasmic components such as pleomorphic mitochondria varying in shape and size, dilated cisternae of RER associated with mitochondria and decreased lysosomes were observed in tumor cells from the NDEA group. Variable light and dark granules of fat and glycogen deposits were also observed. The liver cells from the LycT + NDEA group showed mild damage to the nuclear membrane with fewer karyotin deposits (Figure 2F). Hepatocytes were evident with prominent nucleoli, pleomorphic mitochondria and a higher number of lysosomes. Moreover, liver sections showed many macrophages in sinusoids and apoptotic cells (Figure 2G-H). Table 3 shows the results of a quantitative comparison of hepatic tissues from the NDEA and LycT + NDEA groups using TEM.

Figure 2
Figure 2 Transmission electron micrographs (× 2550) of liver tissue. A and B: Control group illustrating round nucleus (N), nuclear membrane (Nm), nucleolus (Nu), and chromatin (C) and nucleoplasm (Np). Clear cytoplasm with mitochondria (M), dark bodies as lysosomes (Ly), smooth endoplasmic reticulum (SER), rough endoplasmic reticulum (RER), and Ggolgi bodies (GB) surrounded by plasma membrane (PM) were observed; C: LycT group illustrating similar characteristics to those in theof control liver micrograph; D and E: NDEA group illustrating deformed N with disrupted and convoluted Nm, darkly masses in the nucleolus (white arrows), deposition of karyotin (k) and pseudo-inclusions (Pi). Cytoplasm was also found to be compactly packed with multiple pleomorphic M and RER. Percentage of other organelles was found to be lower, and variable light and dark granules of fat (encircled) and glycogen (Gy) deposits were also observed; F-H: LycT + NDEA group illustrating perforated nuclear membrane. Cytoplasm containedwas occupied with pleomorphic mitochondria, with ahowever higher percentage of organelles such as GB, Ly, RER and higher glycogen deposits. Macrophages in the sinusoid (S) were characterized by a large nucleus to cytoplasmic ratio, abundant mitochondria and microvilli (Mi). Apoptotic body characterized by cell shrinkage and condensation of nuclear chromatin into delineated masses (Cm), forming extensions (Ex), and crowded with closely packed cellular organelles (white arrow) and fragments of nucleus (Fr). LycT: Lycopene extracted from tomatoes; NDEA: N-Nitrosodiethylamine.
Table 3 Comparative analysis of the N-Nitrosodiethylamine and lycopene extracted from tomatoes + N-Nitrosodiethylamine group by transmission electron microscopy.
Groups/parametersNDEALycT + NDEA
HepatocytesRounded, smaller in sizePolygonal but rounded edges
NucleusLarge and irregular shapeOval shaped
Nuclear membraneNot uniform and pseudoinclusionsNot uniform
NucleoliLarge, irregular, multipleOne-two
Nuclear/cytoplasmic ratioHighLow
Karyotin (reticular material)Deposition along nuclear membraneFewer karyotin deposits
CytoplasmLoss of organization, denseOrganized
MitochondriaPleomorphic with increased densityPleomorphic but number less than that in the NDEA group
LysosomesFew in numberHigh in number
Fat and glycogen globulesVariableHigh
MacrophagesVery fewMany
Apoptotic bodiesNoClearly visible in the section

A significant increase in the activities of liver hexokinase, PGI and aldolase in the NDEA and LycT + NDEA groups was evident when compared to the control and LycT groups. However, LycT pre-treatment significantly lowered the activity of hexokinase and PGI in the LycT + NDEA group when compared to the NDEA group. No significant change in aldolase level was observed in the LycT + NDEA group when compared to the NDEA group (Figure 3A-C). Moreover, NDEA treatment caused a significant increase in liver G6PD activity in the NDEA group when compared to the control and LycT groups. Moreover, a significant increase was also observed in the levels of liver G6PD in the LycT + NDEA group when compared to the control group. However, G6PD level following LycT pre-treatment in NDEA-challenged mice was observed to be significantly lower than that in the NDEA group. No significant change was observed in the level of G6PD in the LycT group when compared to the control group (Figure 3D). A significant decrease in liver glycogen level was observed in the NDEA group when compared to the control and LycT groups. LycT pre-treatment in NDEA-challenged mice caused a significant increase in the levels of tissue glycogen when compared to the NDEA group. However, a significant decrease in tissue glycogen level was observed when compared to the control and LycT groups (Figure 3E). No significant change was observed in the activities of these enzymes and the level of glycogen in the LycT group when compared to the control group.

Figure 3
Figure 3 Effect of lycopene extracted from tomatoes and/or N-Nitrosodiethylamine. A: Hexokinase activity (nmoles of pseudo-inclusions liberated/min per milligram protein); B: Phosphoglucoisomerase (nmoles of fructose liberated/min per milligram protein); C: Aldolase (μmoles of hydrazone formed/min per milligram protein); D: G6PD (μmoles of NADPH formed/min per milligram protein); E: Glycogen (mg glucose/100 mg liver). aP ≤ 0.05, compared to the control group; b,c,dP ≤ 0.001, compared to the control group, NDEA group and LycT group, respectively; e,fP ≤ 0.01, compared to the control group and LycT group, respectively. LycT: Lycopene extracted from tomatoes; NDEA: N-Nitrosodiethylamine.

Figure 4 shows the mRNA expression of various genes involved in proliferation during HCC in the different treatment groups. Densitometric analysis of HIF-1α expression revealed a significant increase in the NDEA and LycT + NDEA groups when compared to the control and LycT groups (Figure 4). A significant increase in the expression of PCNA and Cyclin D1 was observed in the NDEA group when compared to the control and LycT groups. The LycT + NDEA group showed a significant decrease in mRNA expression of PCNA and Cyclin D1 when compared to the NDEA group. A significant increase in the expression of Cyclin D1 was observed in the LycT + NDEA group when compared to the control and LycT groups. Densitometric analysis of p21 expression revealed a significant decrease in the NDEA group when compared to the control and LycT groups. A significant increase in the expression of p21 was observed in the LycT + NDEA group when compared to the control, NDEA and LycT groups. No change in the expression of these genes was observed when the LycT group was compared to the control group.

Figure 4
Figure 4 Effect of lycopene extracted from tomatoes on mRNA expression of PCNA, Cyclin D1, p21 and HIF-1α during N-Nitrosodiethylamine - induced hepatocarcinogenesis in mice. a,b,cP ≤ 0.001, compared to the N-Nitrosodiethylamine group, LycT group and control group, respectively. LycT: Lycopene extracted from tomatoes; NDEA: N-Nitrosodiethylamine.

Table 4 shows the protein expression of various genes related to proliferation during HCC in the different treatment groups. The expression of HIF-1α was significantly increased in the NDEA and LycT + NDEA groups when compared to the control and LycT groups. Significantly enhanced expression of PCNA and Cyclin D1 was attributed to significantly higher absorbance at 405 nm in the NDEA group when compared to the control and LycT groups (Table 4). A significantly lower absorbance at 405 nm was observed for PCNA expression following LycT administration in NDEA-challenged mice when compared to the NDEA group. However, significantly enhanced expression of PCNA and Cyclin D1 was observed in the LycT + NDEA group when compared to the control and LycT groups. Protein expression of p21 was analyzed using ELISA in all treatment groups (Table 4). A significantly lower absorbance at 405 nm was found in the NDEA group when compared to the control and LycT groups. Significantly increased expression of p21 was observed following LycT pre-treatment in NDEA-challenged mice when compared to the NDEA group. No significant change in the expression of HIF-1α, PCNA, Cyclin D1, and p21 was observed between the LycT and control groups.

Table 4 Effect of N-Nitrosodiethylamine and/or lycopene extracted from tomatoes on protein expression in mice hepatic tissue using ELISA.
Protein (absorbance at 405 nm)ControlNDEALycTLycT + NDEA
PCNA0.31 ± 0.010.47 ± 0.01a0.31 ± 0.01b  0.39 ± 0.01a,b,c
Cyclin D10.25 ± 0.020.38 ± 0.02a0.24 ± 0.02b 0.36 ± 0.01a,c
p210.40 ±0.020.33 ± 0.02e0.39 ± 0.02f0.37 ± 0.01g
HIF-1α0.22 ± 0.030.41 ± 0.01a0.23 ± 0.03b  0.36 ± 0.04a,c
DISCUSSION

Previously, we observed that LycT yielded lycopene phyto-complex (LycT) which delayed and reduced the severity of NDEA-induced HCC as indicated by histopathology, tumor statistics and antioxidant defence system analysis[21]. The presence of a myriad number of compounds in the extract has been reported to enhance the medicinal properties of active components through synergistic effects[29,30]. The therapeutic activity of a medicinal plant is not due to a single component or a few components. However, one substance is so dependent on the presence of another substance that the plant or part of the plant when used in its entirety often yields better results than any single component if used in isolation. Lycopene extraction following basic solvent separation has been proved to be a better agent as there is substantial evidence to show that synergism further enhances its activity and efficacy[31]. Lycopene phyto-complex has also shown high efficacy in triggering apoptosis in addition to its anti-oxidative property[31]. However, the study would be incomplete if the effects of LycT on other hallmark pathways of HCC development were not demonstrated. One of the essential and necessary alterations for the development of almost all cancers is the induction of aerobic glycolysis (Warburg effect). Recently, scientists have linked sustained aerobic glycolysis to oncogenic mutations leading to abnormal cell proliferation and apoptosis[32]. Limited literature is available regarding the irreversible alterations in hepatic architecture during HCC development and their association with other dysregulated carcinogenic insults. With this in mind, the present study was designed to provide an insight into the alterations in ultrastructure, cell proliferation and aerobic glycolysis in NDEA-induced HCC and the effects of LycT on NDEA-induced HCC.

Several irreversible distortions using SEM and TEM were clearly observed and indicated the transformation of well-differentiated HCC to undifferentiated HCC in the NDEA group. Rapidly dividing tumor cells attaining a round contour during crowding of the cells has been reported in the literature[33]. Gross changes in nuclear morphology, epigenetic regulation, chromatin packing and overall nuclear architecture can be related to alterations in the molecular machinery[34]. However, LycT pre-treatment in NDEA-challenged mice resulted in reduced severity as depicted in micrographs. Increased lysosomal bodies and the presence of apoptotic bodies in hepatic tissue from the LycT + NDEA treated group revealed a high apoptotic rate and thus confirms the observations and strengthened our reported data. Aerobic glycolysis arises as a compensatory mechanism due to altered respiration in tumor cells to fulfil the ATP requirements during abnormal cell proliferation[35]. Similarly, enhanced hepatic hexokinase, PGI and aldolase activities in the NDEA group can be attributed to the high cell density observed. Such observations are in accordance with the available literature where enhanced glycolytic enzymes have been linked with chemically induced HCC[36]. Elevated levels of PGI have recently emerged as an excellent response to cancer and PGI level is used as a marker of metastatic growth in patients[37]. Moreover, significant reductions in the activities of glycolytic enzymes following LycT pre-treatment in NDEA-challenged mice were inversely related to HCC development. Histopathological and ultrastructural observations revealed well-differentiated HCC in the LycT + NDEA group, whereas poorly to undifferentiated HCC was observed in the NDEA group[21]. These structural observations could be correlated with the observed modulation of the glycolytic pathway. Various studies based on 18F-FDG uptake on PET scans in different HCCs revealed that well differentiated HCC showed lower 18F-FDG uptake in comparison with poorly differentiated HCC[38].

Ectopic expression of G6PD promotes the survival of tumor cells by maintaining both extracellular pH and redox potential[39]. Moreover, loss of p53 or mutated p53 has been linked with enhanced glucose consumption via increased activity of G6PD[40]. In the current study, LycT pre-treatment in NDEA-challenged mice resulted in significantly low expression of G6PD indicating the inhibitory role of lycopene in HCC by affecting metabolic pathways. Although there is limited research on the role of lycopene in regulating G6PD, some researchers have demonstrated the inhibitory effect of phytochemicals by regulating the activity of G6PD[41]. Decreased expression of Bcl-2 and enhanced p53 expression in the LycT + NDEA group may be responsible for reduced G6PD activity. In the current study, liver glycogen content in HCC was found to be decreased in the NDEA group. However, the reasons for the lack of glycogen accumulation were not fully explored. The transformation of liver cells to tumor cells causes a loss of glucose production via gluconeogenesis. According to the literature, overproduction of the molecule, microRNA-23a, is responsible for inhibiting gluconeogenesis[42]. Glycogen metabolism then acts as an alternate energy source, enabling growth of the cell under metabolic stress. A significant increase in the level of glycogen content was observed in the LycT + NDEA group when compared to the NDEA group. The current observations indicate that lycopene may interfere with glycogen conversion to glucose or might be due to less severe early HCC followed by lower ATP requirement. Such observations are in accordance with the previously reported effect of lycopene in CCL4-challenged mice[43].

These observations in the NDEA group clearly point to a high rate of cell proliferation, which was further evident from increased PCNA and Cyclin D1 expression. Enhanced proliferation attributed to increased expression of PCNA and Cyclin D1 has been reported in the literature[44]. Decreased expression of p21 in the NDEA group could be correlated with enhanced expression of Cyclin D1 as p21 is known to inhibit the activity of cyclin-CDKs complexes[45]. p21 is an important downstream mediator of p53 and its anti-proliferative property plays an important role in preventing tumor development. In our previous study it was observed that reduced p53 expression in the NDEA group was correlated with evasion of apoptosis[22]. Moreover, the LycT + NDEA group showed significantly enhanced expression of p21 and reduced expression of PCNA when compared to the NDEA group indicating the anti-proliferative activity of LycT. Although it is difficult to comment on how lycopene or its metabolites inhibit HCC, the literature shows that treatment with lycopene or its metabolite increased p21 expression and hence aided in preventing NDEA-induced cancer[46]. This observation is also supported by the current observation of increased cell density as shown by SEM and the hepatic glycolysis rate. Moreover, enhanced mRNA and protein expression of HIF-1α clearly indicated the existence of hypoxic conditions in NDEA-treated liver tissue. Various research groups have observed that the expression of HIF-1 modulates apoptosis in HCC[11]. Reports have demonstrated that hypoxia enhances VEGF expression and decreases the ratio of Bax/Bcl-2, thus blocking apoptosis[47]. Pre-treatment with LycT in NDEA-challenged mice resulted in a significant reduction in the expression of HIF-1α at week 24 when compared to the NDEA group. Many reports have demonstrated similar observations where lycopene had an inhibitory response on HIF-1 in both in vivo and in vitro studies. Upadhyay et al[48] performed a comparative study of different antioxidants in order to assess their cancer preventive activity through the inhibition of HIF-1 activity. According to the results, HIF-1α operated in the presence of free radicals and antioxidants with maximum scavenging efficiency for ROS cause inhibition of HIF-1α[49]. The literature also supports the consumption of tomatoes and lycopene mostly inhibited the expression of HIF-1α during prostate carcinogenesis[50]. Such reports strengthen our current observations, that the delay in HCC development may be attributed to the anti-proliferative effect of lycopene. In summary, our report demonstrates the potential of lycopene as a multi-targeted approach against chemically induced HCC.

Data from the present study and previously published studies show that LycT has beneficial effects against NDEA-induced HCC. Electron micrographs (SEM and TEM) of liver biopsies from the different treatment groups provided a picture of 3-D in vivo tissue modulations and thus served as an efficient and accurate tool for demonstrating carcinogenesis and the efficacy of chemopreventive agents along with histopathological observations. Moreover, aerobic glycolysis is also a potential target for determining the chemopreventive efficacy of lycopene and other phytochemicals in preventing carcinogenesis. As shown in the present study, LycT pre-treatment ameliorated disturbed metabolism, however, further studies are warranted to understand the in-depth pharmacokinetics and pharmacodynamics related to lycopene in experimental models of cancer. Finally, an attempt was made to represent diagrammatically the anti-carcinogenic effect of lycopene based on the current study and previous publications (Figure 5).

Figure 5
Figure 5 Overall mechanism of lycopene extracted from tomatoes mediated chemoprevention. LycT: Lycopene extracted from tomatoes; NDEA: N-Nitrosodiethylamine; HCC: Hepatocellular carcinoma; G6PD: Glucose-6-phosphate dehydrogenase.
ACKNOWLEDGMENTS

Financial assistance provided by the University Grant Commission, New Delhi, (India) to carry out the present research is gratefully acknowledged.

COMMENTS
Background

Cancer is a complex disease and requires attention in multiple directions to prevent its development. Among the various carcinogenic insults, uncontrolled proliferation, dysregulated carbohydrate metabolism and hypoxia play very crucial roles in the development of hepatocellular carcinoma (HCC). Although there have been advances in therapeutic approaches a complete cure is still unavailable. Dietary multi-targeted agents have attracted the attention of cancer biologists as these agents may provide a solution to this complex problem by targeting multiple targets simultaneously. Lycopene, a polyunsaturated hydrocarbon imparting red colour to various fruits, is a nutritionally important carotenoid exhibiting beneficial health effects due to its antioxidant activity with minimal side effects. In addition to its antioxidant property, lycopene is known to modulate other non-oxidative pathways such as regulation of gap junction communication, the hormonal system, immune system and metabolic pathways of xenobiotics. Previously, we demonstrated that lycopene from tomatoes is a potent agent for inhibiting HCC development in terms of histopathological observations, tumor statistics, apoptosis, antioxidative capacity and toxicity. However, studies are warranted to determine the modulating effect of lycopene on dysregulated glucose metabolism and hypoxia, as these processes play critical roles in cancer. Thus, in the current study, the influence of lycopene extracted from tomatoes on various glycolytic and non-glycolytic enzymes, the expression of hypoxia inducible factor-1α (HIF-1α) and potent cell proliferation-associated genes while preventing N-Nitrosodiethylamine (NDEA)-induced HCC was investigated. Moreover, an attempt was made to demonstrate the impact of an imbalance between energy production and metabolic demands on the gross morphology and ultrastructure of hepatocytes in HCC.

Research frontiers

Important areas related to the current study include: (1) carcinogenesis: Incidence rate, statistics, prognosis, consequences, mortality, molecular and biochemical markers, altered cellular pathways and therapeutic limitations; and (2) chemoprevention: Natural agents, multifaceted approach, lycopene a colored pigment and its antioxidative and anti-carcinogenic potential.

Innovations and breakthroughs

Despite significant research efforts, cancer is considered an incurable disease due to its high incidence rate, poor prognosis, high mortality rate, multifactorial causative agents, and side effects associated with chemotherapeutics. As natural agents are safer and have a multifaceted approach they are considered an alternative therapy. Lycopene is known to be a potent antioxidant and phytoagent with a protective effect against chronic diseases such as cancer. However, the detailed mechanism underlying its anti-carcinogenic effects is unclear. The basic requirement in the present investigation was to design a study that could demonstrate the action of lycopene at various stages to cover the maximum number of carcinogenic bioprocesses. The present study is part of this research design, in which hepatic tissue from different groups, i.e., control, LycT, NDEA and LycT + NDEA groups was studied at different levels including structural markers, electro-physical markers, morphological markers, biochemical markers, and molecular markers that are known to be involved in the development of cancer. This type of study provides innovation and achievement for futuristic analyses of natural agents in disease models.

Applications

The outcomes of the current study provide deeper insight into the mechanistic targets of lycopene which shows ameliorating effects. Lycopene administration directly or indirectly stimulated various molecular targets such as p53, which have been correlated with decreased G6PD activity or biosynthetic pathways that play important roles during tumor proliferation. The protective effect of lycopene can be studied at the ultrastructural level by scanning and transmission electron microscopy (SEM and TEM). A detailed explanation regarding hepatic SEM and TEM during carcinogenesis is also information which is ambiguous in the literature.

Peer-review

This manuscript entitled "Lycopene modulates cellular proliferation, glycolysis and hepatic ultrastructure during hepatocellular carcinoma" investigated the effect of lycopene on ultra-structure, glycolytic enzymes, cell proliferation markers and hypoxia during NDEA induced hepatocarcinogenesis.

Footnotes

Manuscript source: Invited manuscript

Specialty type: Gastroenterology and hepatology

Country of origin: India

Peer-review report classification

Grade A (Excellent): 0

Grade B (Very good): 0

Grade C (Good): C

Grade D (Fair): D

Grade E (Poor): 0

P- Reviewer: Camacho J, Xu MY S- Editor: Qi Y L- Editor: Webster JR E- Editor: Li D

References
1.  Anand P, Kunnumakkara AB, Sundaram C, Harikumar KB, Tharakan ST, Lai OS, Sung B, Aggarwal BB. Cancer is a preventable disease that requires major lifestyle changes. Pharm Res. 2008;25:2097-2116.  [PubMed]  [DOI]
2.  Theodoratou E, Farrington SM, Tenesa A, McNeill G, Cetnarskyj R, Korakakis E, Din FV, Porteous ME, Dunlop MG, Campbell H. Associations between dietary and lifestyle risk factors and colorectal cancer in the Scottish population. Eur J Cancer Prev. 2014;23:8-17.  [PubMed]  [DOI]
3.  Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray F. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 2015;136:E359-E386.  [PubMed]  [DOI]
4.  Inami K, Ishikawa S, Mochizuki M. Activation mechanism of N-Nitrosodialkylamines as environmental mutagens and its application to antitumor research. Gene Environ. 2009;31:97-104.  [PubMed]  [DOI]
5.  Bharati S, Rishi P, Koul A. Azadirachta indica exhibits chemopreventive action against hepatic cancer: Studies on associated histopathological and ultrastructural changes. Microsc Res Tech. 2012;75:586-595.  [PubMed]  [DOI]
6.  Crews C. The determination of N-nitrosamines in food. Qual Assur Saf Crop. 2010;2:2-12.  [PubMed]  [DOI]
7.  Heindryckx F, Colle I, Van Vlierberghe H. Experimental mouse models for hepatocellular carcinoma research. Int J Exp Pathol. 2009;90:367-386.  [PubMed]  [DOI]
8.  Aiub CA, Gadermaier G, Ferreira F, Felzenszwalb I, Eckl P, Pinto LFR. N-Nitrosodiethylamine cytochrome P450 induction and cytotoxicity evaluation in primary cultures of rat hepatocytes. Am J Mol Biol. 2011;1:70-78.  [PubMed]  [DOI]
9.  Aiub CA, Gadermaier G, Silva IO, Felzenszwalb I, Pinto LF, Ferreira F, Eckl P. N-nitrosodiethylamine genotoxicity evaluation: a cytochrome P450 induction study in rat hepatocytes. Genet Mol Res. 2011;10:2340-2348.  [PubMed]  [DOI]
10.  Ortega AD, Sánchez-Aragó M, Giner-Sánchez D, Sánchez-Cenizo L, Willers I, Cuezva JM. Glucose avidity of carcinomas. Cancer Lett. 2009;276:125-135.  [PubMed]  [DOI]
11.  Semenza GL. Hypoxia and cancer. Cancer Metastasis Rev. 2007;26:223-224.  [PubMed]  [DOI]
12.  Wu X, Patterson S, Hawk E. Chemoprevention--history and general principles. Best Pract Res Clin Gastroenterol. 2011;25:445-459.  [PubMed]  [DOI]
13.  Mehta RG, Murillo G, Naithani R, Peng X. Cancer chemoprevention by natural products: how far have we come? Pharm Res. 2010;27:950-961.  [PubMed]  [DOI]
14.  Singh S, Singh PP, Roberts AR, Sanchez W. Chemopreventive strategies in hepatocellular carcinoma. Nat Rev Gastroenterol Hepatol. 2014;11:45-54.  [PubMed]  [DOI]
15.  Seren S, Lieberman R, Bayraktar UD, Heath E, Sahin K, Andic F, Kucuk O. Lycopene in cancer prevention and treatment. Am J Ther. 2008;15:66-81.  [PubMed]  [DOI]
16.  Heber D, Lu QY. Overview of mechanisms of action of lycopene. Exp Biol Med (Maywood). 2002;227:920-923.  [PubMed]  [DOI]
17.  Coyne T, Ibiebele TI, Baade PD, Dobson A, McClintock C, Dunn S, Leonard D, Shaw J. Diabetes mellitus and serum carotenoids: findings of a population-based study in Queensland, Australia. Am J Clin Nutr. 2005;82:685-693.  [PubMed]  [DOI]
18.  Mariani S, Lionetto L, Cavallari M, Tubaro A, Rasio D, De Nunzio C, Hong GM, Borro M, Simmaco M. Low prostate concentration of lycopene is associated with development of prostate cancer in patients with high-grade prostatic intraepithelial neoplasia. Int J Mol Sci. 2014;15:1433-1440.  [PubMed]  [DOI]
19.  Liu CL, Huang YS, Hosokawa M, Miyashita K, Hu ML. Inhibition of proliferation of a hepatoma cell line by fucoxanthin in relation to cell cycle arrest and enhanced gap junctional intercellular communication. Chem Biol Interact. 2009;182:165-172.  [PubMed]  [DOI]
20.  Goyal PK. Cancer chemoprevention by natural products: current & future prospects. J Integr Oncol. 2012;1:e101.  [PubMed]  [DOI]
21.  Gupta P, Bansal MP, Koul A. Spectroscopic characterization of lycopene extract from Lycopersicum esculentum (Tomato) and its evaluation as a chemopreventive agent against experimental hepatocarcinogenesis in mice. Phytother Res. 2013;27:448-456.  [PubMed]  [DOI]
22.  Gupta P, Bansal MP, Koul A. Evaluating the effect of lycopene from Lycopersicum esculentum on apoptosis during NDEA induced hepatocarcinogenesis. Biochem Biophys Res Commun. 2013;434:479-485.  [PubMed]  [DOI]
23.  Gupta P, Bansal MP, Koul A. Lycopene modulates initiation of N-nitrosodiethylamine induced hepatocarcinogenesis: studies on chromosomal abnormalities, membrane fluidity and antioxidant defense system. Chem Biol Interact. 2013;206:364-374.  [PubMed]  [DOI]
24.  Crane RK, Sols A. Animal tissue hexokinases: (soluble and particulate forms). Meth Enzymol. 1955;1:277-286.  [PubMed]  [DOI]
25.  Jagannathan V, Singh K, Damodaran M. Carbohydrate metabolism in citric acid fermentation: Purification and properties of aldolase from Aspergillus niger. Biochem J. 1956;63:94-105.  [PubMed]  [DOI]
26.  Horrocks JE, Ward J, King J. A routine method for the determination of phosphoglucose isomerase activity in body fluid. J Clin Pathol. 1963;16:248-251.  [PubMed]  [DOI]
27.  Seifter S, Dayton S. The estimation of glycogen with the anthrone reagent. Arch Biochem. 1950;25:191-200.  [PubMed]  [DOI]
28.  Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265-275.  [PubMed]  [DOI]
29.  Stahl W, Sies H. Bioactivity and protective effects of natural carotenoids. Biochim Biophys Acta. 2005;1740:101-107.  [PubMed]  [DOI]
30.  Chandra S, Sah K, Bagewadi A, Keluskar V, Shetty A, Ammanagi R, Naik Z. Additive and synergistic effect of phytochemicals in prevention of oral cancer. Eur J of Gen Pract. 2012;1:142-147.  [PubMed]  [DOI]
31.  Shi J, Qu Q, Kakùda Y, Yeung D, Jiang Y. Stability and synergistic effect of antioxidative properties of lycopene and other active components. Crit Rev Food Sci Nutr. 2004;44:559-573.  [PubMed]  [DOI]
32.  Cairns RA, Harris IS, Mak TW. Regulation of cancer cell metabolism. Nat Rev Cancer. 2011;11:85-95.  [PubMed]  [DOI]
33.  Paxton JR, Bolger BS, Armour A, Symonds RP, Mao JH, Burnett RA. Apoptosis in cervical squamous carcinoma: predictive value for survival following radiotherapy. J Clin Pathol. 2000;53:197-200.  [PubMed]  [DOI]
34.  Di Micco R, Sulli G, Dobreva M, Liontos M, Botrugno OA, Gargiulo G, dal Zuffo R, Matti V, d'Ario G, Montani E. Interplay between oncogene-induced DNA damage response and heterochromatin in senescence and cancer. Nat Cell Biol. 2011;13:292-302.  [PubMed]  [DOI]
35.  Warburg O. On respiratory impairment in cancer cells. Science. 1956;124:269-270.  [PubMed]  [DOI]
36.  Langeswaran K, Revathy R, Kumar SG, Vijayaprakash S, Balasubramanian MP. Kaempferol ameliorates aflatoxin B1 (AFB1) induced hepatocellular carcinoma through modifying metabolizing enzymes membrane bound ATPases and mitochondrial TCA cycle enzymes. Asian Pac J Trop Biomed. 2012;2:S1653-S1659.  [PubMed]  [DOI]
37.  Tsutsumi S, Fukasawa T, Yamauchi H, Kato T, Kigure W, Morita H, Asao T, Kuwano H. Phosphoglucose isomerase enhances colorectal cancer metastasis. Int J Oncol. 2009;35:1117-1121.  [PubMed]  [DOI]
38.  Lee JD, Yang WI, Park YN, Kim KS, Choi JS, Yun M, Ko D, Kim TS, Cho AE, Kim HM. Different glucose uptake and glycolytic mechanisms between hepatocellular carcinoma and intrahepatic mass-forming cholangiocarcinoma with increased (18)F-FDG uptake. J Nucl Med. 2005;46:1753-1759.  [PubMed]  [DOI]
39.  Kobayashi M, Fujita I, Itagaki S, Hirano T, Iseki K. Transport mechanism for L-lactic acid in human myocytes using human prototypic embryonal rhabdomyosarcoma cell line (RD cells). Biol Pharm Bull. 2005;28:1197-1201.  [PubMed]  [DOI]
40.  Jiang P, Du W, Wang X, Mancuso A, Gao X, Wu M, Yang X. p53 regulates biosynthesis through direct inactivation of glucose-6-phosphate dehydrogenase. Nat Cell Biol. 2011;13:310-316.  [PubMed]  [DOI]
41.  Edderkaoui M, Hui H, Li G, Xu J, Lee WN, Go VLW, Pandol SJ. Phytochemicals inhibit proliferation and promote death through NADPH oxidase and G6PD. Cancer Res. 2010;70 (Suppl 8) Abstract Nr 1895.  [PubMed]  [DOI]
42.  Wang XD. Lycopene metabolism and its biological significance. Am J Clin Nutr. 2012;96:1214S-1222S.  [PubMed]  [DOI]
43.  Ebeid HM, Gibriel AA, Al-Sayed HM, Elbehairy SA, Motawe EH. Hepatoprotective and antioxidant effects of wheat, carrot, and mango as nutraceutical agents against CCl4-induced hepatocellular toxicity. J Am Coll Nutr. 2015;34:228-231.  [PubMed]  [DOI]
44.  Stoimenov I, Helleday T. PCNA on the crossroad of cancer. Biochem Soc Trans. 2009;37:605-613.  [PubMed]  [DOI]
45.  Perkins ND. Not just a CDK inhibitor: regulation of transcription by p21(WAF1/CIP1/SDI1). Cell Cycle. 2002;1:39-41.  [PubMed]  [DOI]
46.  Ip BC, Hu KQ, Liu C, Smith DE, Obin MS, Ausman LM, Wang XD. Lycopene metabolite, apo-10'-lycopenoic acid, inhibits diethylnitrosamine-initiated, high fat diet-promoted hepatic inflammation and tumorigenesis in mice. Cancer Prev Res (Phila). 2013;6:1304-1316.  [PubMed]  [DOI]
47.  Baek JH, Jang JE, Kang CM, Chung HY, Kim ND, Kim KW. Hypoxia-induced VEGF enhances tumor survivability via suppression of serum deprivation-induced apoptosis. Oncogene. 2000;19:4621-4631.  [PubMed]  [DOI]
48.  Upadhyay J, Kesharwani R. K, Misra K. Comparative study of antioxidants as cancer preventives through inhibition of HIF-1 alpha activity. Bioinformation. 2009;4:233-236.  [PubMed]  [DOI]
49.  Burlaka AP, Sidorik EP, Ganusevich II, Osinsky SP. Effects of radical oxygen species and NO: formation of intracellular hypoxia and activation of matrix metalloproteinases in tumor tissues. Exp Oncol. 2006;28:49-53.  [PubMed]  [DOI]
50.  Thomas-Ahner JM, Wan L, Tan HL, Moran NE, Elsen AC, Pearl DK, Erdman JW, Clinton SK. Tomato carotenoids and testosterone modulate mRNA and miRNA profiles during prostate carcinogenesis. Cancer Res. 2013;73:Abstract Nr. 3701.  [PubMed]  [DOI]