Search Article Keyword:  



PubMed Submission Abstract PDF Feed Back  Click Count: 5242 DownLoad Count: 2014 



ISSN 1007-9327 CN 14-1219/R  World J Gastroenterol  2007 October 28; 13(40): 5299-5305

Effects and mechanisms of silibinin on human hepatoma cell lines

John J Lah, Wei Cui, Ke-Qin Hu





John J Lah, Wei Cui, Ke-Qin Hu, Division of Gastroenterology, University of California, Irvine Medical Center, 101 The City Drive, Building 53, Suite 113, Orange, CA 92868, United States

Supported by UCI institutional research grants from GI Division and Chao Family Comprehensive Cancer Center (K.-Q.H.)

Correspondence to: Ke-Qin Hu, Division of Gastroenterology, University of California, Irvine Medical Center, 101 The City Drive, Building 53, Suite 113, Orange, CA 92868, United States.

Telephone: +1-714-4566745  Fax: +1-714-4567753

Received: April 7, 2007         Revised: July 25, 2007



AIM: To investigate in vitro effects and mechanisms of silibinin on hepatocellular carcinoma (HCC) cell growth.


METHODS: Human HCC cell lines were treated with different doses of silibinin. The effects of silibinin on HCC cell growth and proliferation, apoptosis, cell cycle progression, histone acetylation, and other related signal transductions were systematically examined.


RESULTS: We demonstrated that silibinin significantly reduced the growth of HuH7, HepG2, Hep3B, and PLC/PRF/5 human hepatoma cells. Silibinin-reduced HuH7 cell growth was associated with significantly up-regulated p21/CDK4 and p27/CDK4 complexes, down-regulated Rb-phosphorylation and E2F1/DP1 complex. Silibinin promoted apoptosis of HuH7 cells that was associated with down-regulated survivin and up-regulated activated caspase-3 and -9. Silibinin's anti-angiogenic effects were indicated by down-regulated metalloproteinase-2 (MMP2) and CD34. We found that silibinin-reduced growth of HuH7 cells was associated with increased activity of phosphatase and tensin homolog deleted on chromosome ten (PTEN) and decreased p-Akt production, indicating the role of PTEN/PI3K/Akt pathway in silibinin-mediated anti-HCC effects. We also demonstrated that silibinin increased acetylation of histone H3 and H4 (AC-H3 and AC-H4), indicating a possible role of altered histone acetylation in silibinin-reduced HCC cell proliferation.


CONCLUSION: Our results defined silibinin's in vitro anti-HCC effects and possible mechanisms, and provided a rationale to further test silibinin for HCC chemoprevention.


2007 WJG. All rights reserved.


Key words: Hepatocellular carcinoma; HuH7 cells; Silibinin; Chemoprevention; Cell cycle; Cell cycle progression; Apoptosis; Acetylation of histone


Lah JJ, Cui W, Hu KQ. Effects and mechanisms of
silibinin on human hepatoma cell lines. World J Gastroenterol 2007; 13(40): 5299-5305



Hepatocellular carcinoma (HCC) is one of the most common malignancies related to a high mortality glo-bally[1,2]. Recent studies have noted a significant rise in the incidence of HCC in the United States in the past 2 decades[2]. Less than 1% of HCC patients underwent a radical surgical resection in the US between 1974 and 1996[3]. HCC's limited treatment remedies and the poor prognosis emphasize the importance in developing an effective chemoprevention for this disease.

Milk thistle (Silybum marianum) has been widely utilized as a folk remedy for liver diseases. It is a popular dietary supplement widely used in the United States and Europe[4]. Silibinin is a polyphenolic flavonoid and the major biologically active compound of milk thistle[4-6]. It is well known that milk thistle is safe and well-tolerated, and it protects the liver from drug or alcohol-related injury[7,8]. Studies demonstrated silibinin's inhibitory effects on multiple cancer cell lines, including prostate[9-12], colon[13,14], skin[15-17], bladder[18,19] and lung cancers[20]. Recently, we and Varghese et al reported silibinin's anti-HCC effects[21,22], but further studies are needed to define silibinin's inhibitory effects and mechanisms on human HCC cell growth.

Searching for non-invasive biomarkers is another important filed of HCC chemoprevention. Plasma alpha-fetoprotein (AFP) has been used as a clinical marker for diagnosing and monitoring recurrent HCC[23-25]. However, AFP's value in monitoring effect of HCC chemoprevention has not been tested before.

Phosphatase and tensin homolog deleted on chromo-some ten (PTEN), phosphatidylinositol 3'-kinase (PI3K) and Akt (PTEN/PI3K/Akt) pathway has been associated with carcinogenesis[26]. Activated PI3K-Akt signaling promotes carcinogenesis[27,28]. PTEN is a negative regulator of PI3K-Akt signaling[29] and one of the most frequently inactivated genes in malignancies[30, 31]. Akt is a downstream protein kinase of PI3K (PTEN) and is a signal transduction protein that has been identified as one of the key elements in protecting cells from apoptosis. If unregulated, Akt promotes uncontrolled cell replication[32,33]. It was reported that silibinin affects Akt expression in prostate cancer cells[16], but it remains unknown whether silibinin affects HCC growth through a PTEN/PI3K/Akt pathway in human liver cancer cells.

Histone acetylation modifies nucleosome structure that leads to DNA relaxation, reduces the affinity of histone complexes with DNA, and enhances the access of transcriptional factor to DNA[34]. Accumulating evidence has indicated that alteration of histone acetylation plays an important role in carcinogenesis[35,36], but it remains unknown whether it is associated with silibinin's anti-HCC effects.

In the present study, we demonstrated that silibinin significantly inhibited the growth of HuH7, HepG2, Hep3B, and PLC/PRF/5 human HCC cells that was associated with decreased Ki-67 expression, and cell cycle progression by arresting G1-S transition, and promoted apoptosis. These effects of silibinin were associated with increased PTEN activity and decreased p-Akt production, indicating the role of PTEN/PI3K/Akt pathway in silibinin-mediated anti-HCC effects. We also demonstrated that silibinin increased AC-H3 and AC-H4 expression, indicating that altered histone acetylation is involved in silibinin-reduced HCC cell proliferation.




The cell culture media were the same, as previously reported[37,38]. Anti-activated caspase-3 antibody was purchased from Sigma Chemical Co. (St. Louis, MO). The antibodies against human Ki-67, AFP, p-Rb, E2F1, DP1, CD1, CDK4, p21 and p27, activated caspase-9, bcl-2, survivin, CD34, metalloproteinase (MMP)-2, MMP-9, phosphorylated-AktThr308, PTEN, AC-histone3 and AC-histone4, and b-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). PTEN activity assay kit was from Biomol Research Laboratories, Inc (Plymouth Meeting, PA). An EIA kit for cell death detection was from Roche Applies Science (Indianapolis, IN).


Cell culture

Human HCC cell lines, HuH7, HepG2, PLC/PRF/5, and Hep3B cells[37,38], were used in the present study. All the cells were cultured, as previously reported[37,38]. The experiments were performed when cells reached about 80% confluence and cultured in FBS-free media for 24 h to synchronize the cell growth[37,38].


Cell proliferation assay

Cell proliferation was determined using MTT assay, as previously reported[37,38]. Briefly, the effects of silibinin on HCC cell growth were then determined after 24 h of incubation by optical density absorbance at 490 nm according to the manufacturer's instruction[37,38].


Apoptosis assays

Apoptosis was determined in duplicate using an EIA kit for cell death detection, as previously reported[37,38].


Immunoprecipitation (IP) and immunoblot (IB) assays

After 24 h of treatment with silibinin at 25% inhibitory concentration (IC25) or IC50 dose, the cell pellets were lysed and the supernatants were used to detect Ki-67, AFP, p-Rb, E2F1, CD1, CDK4, p21waf1/cip1, p27kip1, bcl-2, survivin, activated caspase-3 and caspase-9, CD34, MMP-2, MMP-9, phosphorylated-AktThr308, PTEN, and AC-H3 and AC-H4. The IP assays were same, as previously reported[37,38]. b-actin was used as an internal control. The relative amount of each protein was quantified by digitally scanning its hybridizing bands, as previously reported[37,38].


PTEN activity assay

PTEN protein was immunoprecipitated with 10 mL of rabbit anti-human antibody at 4℃ overnight, followed by addition of 25 mL of anti-rabbit IgG-conjugated agarose beads for 2 h at 4℃, washing and centrifugation. The phosphatase reaction was performed in 50 mL of assay buffer containing 200 mmol/L water-soluble diC8-PIP3 and the immunoprecipitated PTEN protein. The release of phosphate from the substrate was measured in a colorimetric assay using the Biomol Green Reagent (Plymouth Meeting, PA)[39]. The OD absorbance at 650 nm was recorded in an ELISA plate reader[37,38].


Statistical analysis

The descriptive statistics was provided with mean SD. A repeated-measure ANOVA test was used to assess dose dependent effects of silibinin on HuH7, HepG2, Hep3B, and PLC/PRF/5 cells. An independent sample t-test was used to assess the effects (i.e. mean differences) of silibinin treatment on apoptosis, and IB results. A P value < 0.05 was considered statistically significant.



Potent dose-dependent anti-proliferative effects of silibinin on human HCC cells

Effects of silibinin were initially assessed in HuH7 cells by MTT assay. As shown in Figure 1A, silibinin resulted in a dose-dependent inhibition of HuH7 cell growth. Compared to the control, there was a dose-dependent inhibitory which became significant at the dose greater than 180 mmol/L (P < 0.05). As shown in Figure 1B, silibinin also significantly inhibited the growth of HepG2, Hep3B, and PLC/PRF/5 human HCC cell lines, indicating a wide spectrum of silibinin's inhibitory effects on human HCC cell growth, as previously reported[21,22]. Because the HuH7 cell line is one of the most commonly used human HCC lines[37,38], it was then used to further determine silibinin's anti-HCC effects and mechanisms. For further characterization of dose-related mechanistic effect of silibinin, approximate IC25 (i.e. 120 mmol/L) and IC50 (i.e. 240 mmol/L) concentration were subsequently used for the remainder of the study.

Ki-67 is a commonly used biomarker for cell prolifera-tion[40]. Consistent with the data derived from MTT as-say, silibinin treatment resulted in a significantly dose-dependent decrease in Ki-67 expression, as shown in Figures 2B (P < 0.05). These data further demonstrated silibinin's significant dose-dependent anti-proliferative effects on human HCC cells.


Effects of silibinin on cell cycle progression

Uncontrolled progression of the cell cycle promotes growth of cancer cells[41]. A major activity of the CD1/CDK4 complex is to initiate phosphorylation of retinoblastoma (Rb) that then fails to maintain it's binding to E2F1, and thus releases the transcription factor to promote cell cycle progression[42]. Previous studies on other cancer cell lines showed a significant inhibitory effect of silibinin on the cell cycle progression[11-13]. In the present study, we found that silibinin resulted in a significant dose-dependent inhibition of CD1/CDK4 complex that was associated with reduced Rb phosphorylation and, E2F1/DP1 complex in HuH7 cells (P < 0.01), as shown in Figure 2C-E.

By binding to the cyclin/CDK complexes, cyclin dependent kinase inhibitors (CDKIs), such as p21 and p27, halt uncontrolled cell proliferation[43]. As noted in previous studies on other cancer cell lines[12-14], we demonstrated that silibinin not only significantly increased p21 and p27 expression (P < 0.01), but also increased formation of p21/CDK4 and p27/CDK4 complexes (Figure 2F-I) in a dose-dependent fashion. Thus, our results demonstrate silibinin inhibits the growth of human hepatoma cells through inhibiting CDK activity.


Effects of silibinin on AFP production and secretion from HuH7 cells

As shown in Figure 3B, compared to untreated HuH7 cells, silibinin at the dose of 120 mmol/L resulted in a significant decrease in AFP production in HuH7 cells (P < 0.05) that was associated with a reduced AFP level in the culture medium (Figure 3C, P < 0.05).


Effects of Silibinin on Apoptosis

Apoptosis is another important regulatory step in contro-lling cancer cell proliferation[4]. Studies indicated that silibinin induces apoptosis in several malignant cell lines[13,14,18], but such effects have not been tested in human hepatoma cells. We demonstrated silibinin dose-dependently increases of apoptosis in HuH7 cells, as shown in Figure 4A (P < 0.01). To understand the mechanisms of silibinin-induced apoptosis, we examined the expression of Bcl-2, survivin, and activated caspase-3 and 9. Our results showed that silibinin-induced apoptosis did not alter bcl-2 expression (data not shown), but resulted in a dose-dependent inhibition of survivin expression (Figure 4C, P < 0.01) that was associated with increased levels of activated caspases-3 and -9 (Figures 4D and 4E, P < 0.01).


Possible effects of silibinin on angiogenesis

In previous studies, silibinin has been reported to inhibit angiogenesis in non-HCC cancer cell lines[13]. To evaluate whether silibinin affects angiogenesis in human HCC cells, we measured the expression of CD34, a transmembrane glycoprotein on vascular cells associated with angiogenesis[44], and MMP-2 and MMP-9, which are markers associated with angiogenesis as well as metastatic invasion[45]. As shown in Figure 5B, silibinin at IC50, but not IC25 dose decreased the expression of CD34 (P < 0.01). At the higher dose, Silibinin also resulted in decrease of MMP-2 (Figure 5C, P < 0.01), but not MMP-9 (data not shown) in HuH7 cells.


Effects of silibinin on PTEN/PI3K/Akt pathway

It has been reported that the PTEN/PI3K/Akt pathway is involved in cancer growth[16,46], As shown in Figure 6B, silibinin-reduced HuH7 cell proliferation was associated with a dose-dependent decrease in p-Akt (P < 0.01) in these cells. PTEN is an upstream negative regulator of Akt. It was reported that altered PTEN expression or activity is associated with the pathogenesis of HCC[47-49]. In the present study, we found that silibinin at IC50 dose did not significantly change PTEN expression (data not shown), but significantly increased PTEN activity (Figure 6E). These results suggested that the PTEN/PI3K/Akt pathway is involved in silibinin-reduced growth of human HCC cells.


Effects of silibinin on AC-H3 and AC-H4 expression

We then examined the association of AC-H3 and AC-H4 expression with silibinin-reduced HCC cell growth. Our results demonstrated that silibinin-reduced HuH7 cell growth was associated with increased AC-H3 and AC-H4 expression (Figures 6 C and D, P < 0.05). These results suggest that increased AC-H3 and AC-H4 expression may play an important role in silibinin-reduced HCC growth.



Searching for an effective chemoprevention of HCC has been an active field of research. Silibinin is a polyphenolic flavonoid and the major biologically active compound of milk thistle. It is well known that milk thistle is safe and well tolerated, and it protects the liver from drug or alcohol-related injury[7,8]. Recent demonstration of silibinin's anti-HCC effects[21,22] provided us with a rationale to further define the related effects and mechanisms of HCC chemoprevention. In the present study, we examined the effects and mechanisms of silibinin on growth of human HCC cells.

Using MTT assay[37,38], we demonstrated that silibinin treatment resulted in a potent inhibition of four different human HCC cell lines, indicating its broad spectrum of anti-HCC effects. We also revealed silibinin's linear dose-dependent inhibition of HuH7 cell growth. Silibinin at IC25 and IC50 doses for HuH7 cells also resulted in reduced growth of HepG2, Hep3B, and PLC/PRF/5 cells, confirming the previous reports[21,22], These results promote us to further test silibinin for HCC chemoprevention.

Both PCNA and Ki-67 are biomarkers for cell proliferation[40]. Singh et al reported silibinin significantly decreases PCNA and Ki-67 expression in nude mice bear-ing xenografts of human prostate cancer[11]. Consistent with this, we have demonstrated that silibinin significantly reduced Ki-67 expression in HuH7 cells in a dose-dependent fashion. These suggest that silibinin reduces growth of human HCC cells by down regulating their proliferation.

AFP is associated with HCC differentiation and has been widely used for diagnosing HCC and assessing treatment effects or recurrence of HCC in humans[24,25]. Our results showed that silibinin treatment resulted in significant decrease in AFP production and secretion that was well correlated with growth inhibition of HuH7 cells. These findings suggest silibinin may promote HCC cell differentiation, and AFP may serve as a non-invasive biomarker to determine silibinin's in vivo anti-HCC effects.

An uncontrolled G1-S progression results in continued proliferation with potential malignant transformation and carcinogenesis. Increased CDK4/CD1 complex enhances Rb phosphorylation that results in release of E2F1 from p-Rb/E2F1 complex and promotes E2F1/DP1 complex formation and stimulates cell cycle progression[42]. Tyagi et al reported that silibinin causes a significant decrease in p-Rb in human prostate cancer cells[10]. Our results indicate that silibinin-inhibited CDK4/CD1 complex formation is one of the important steps that inhibit Rb phosphorylation, followed by reduction of E2F1/DP1 complex formation.

CDKIs are important regulators of the activity of the CD1/CDK4 complex. By binding to the cyclin/CDK complexes, two very important CDKIs, p21 and p27, inhibit their activities. Varghese, et al reported that silibinin increases levels of p27 in human hepatoma cells[22]. Silibinin was also reported increasing expression of p21 in several non-HCC cancer cells[14]. In the present study, we demonstrated that silibinin resulted in a significantly dose-dependent increase in both p21 and p27, which was well correlated with their respective binding to CDK4, the bioactive forms of these CDKIs. Taken together, our data demonstrated that silibinin reduces cell cycle progression in human hepatoma cells by arresting G1-S transition that involves a comprehensive signaling of cell cycle modulators.

Previous data on other cancer cell lines have demon-strated that silibinin has effects on the apoptotic con-trol[10,14,21]. We demonstrated that silibinin causes a signifi-cant increase in apoptosis of HuH7 cells which was asso-ciated with decreased survivin expression and increased activated caspase-3 and -9. Because survivin can bind with caspases[19,50,51], our results suggest that silininin-induced apoptosis of HuH7 cells is mediated by decreased survivin that results in increased caspase-3 and 9 activation.

Angiogenesis is an important aspect of cancer invasion and survival. CD34 is a valuable marker to demonstrate this issue[44]. A previous study demonstrated that silibinin decreases angiogenesis in colon cancer cells[15]. In the present study, we revealed that silibinin at IC50 decreased CD34 protein expression. MMP-2 and MMP-9 have been used as markers for angiogenesis and malignant invasion[45]. It was reported that silibinin resulted in a significant decrease in MMP-2, but not MMP-9 levels in human lung cancer cells[20]. In the present study, we found that silibinin also resulted in a dose-dependent and significant decrease in MMP-2, but not MMP-9 expression. Although our data suggest that silibinin may reduce angiogenesis in human HCC cells, further in vivo studies with quantification of microvessel density[52] will be needed to validate these findings.

There is growing evidence of PTEN/PI3K/Akt path-way in hepatocarcinogenesis[27,32,47-49]. PTEN is a tumor suppressor gene and the deletion or inactivation of this gene has been described in a variety of cancer cell lines[30,33,53]. As a result, the tumor suppressive properties of PTEN relates in part to its ability to down-regulate the Akt pathway and thus inhibit cell proliferation[32,33]. Paramio et al showed that PTEN decreases p-Rb and resulted in down-regulation of CD1[54]. Furthermore, Weng et al demonstrated through the use of breast cancer cells that PTEN also up-regulates p27 and down-regulates CD1[55]. However, it remains to be determined whether PTEN/PI3K/Akt pathway is involved in silibinin-reduced growth of cancers. In the present study, we found that silibinin significantly increased PTEN activity in association with decreased p-Akt in HuH7 cells. Since silibinin treatment also resulted in significant decrease of p-Rb and proliferation in these cells, it is evident that silibinin alters PTEN activity to assist in cellular growth control through downstream regulation of Akt and also possibly in promoting the up-regulation of p27 and the down-regulation of both p-Rb and CD1 as suggested by previous studies[54]. It was also reported that overexpression of PTEN reduces survivin expression[56]. We found that silibinin-mediated increase in PTEN activity and decrease in p-Akt was associated with decreased survivin expression and enhanced apoptosis in HuH7 cells. These data support the notion that PTEN/PI3K/Akt pathway may mediate cancer cell apoptosis by modulating surviving expression, and silibinin may play an important role in this interaction. Additional studies will be needed to further detail the role of PTEN/PI3K/Akt signaling in silibinin-reduced growth of human HCC cells.

Histone acetylation alters chromatin conformation by making promoter regions more accessible to transcription factors and permissive to transcriptional activation[34]. Studies have reported that histone acetylation is involved in cell proliferation, differentiation, and cell cycle regulation[34]. Decrease in acetylation status in the cell is associated with carcinogenesis[35,36]. Our results demonstrated that silibinin-reduced HuH7 cell growth was significantly associated with increased AC-H3 and AC-H4 expression, suggesting that increased histone acetylation may mediate silibinin-reduced HCC growth. Our findings not only indicate silibinin's novel anti-cancer mechanisms, but also provide additional targets for searching new agents for HCC chemoprevention.



1      Chen CJ, Yu MW, Liaw YF. Epidemiological characteristics and risk factors of hepatocellular carcinoma. J Gastroenterol
1997; 12: S294-S308   PubMed

2      El-Serag HB, Mason AC. Rising incidence of hepatocellular carcinoma in the United States. N Engl J Med 1999; 340:
        745-750   PubMed

3      El-Serag HB, Mason AC, Key C. Trends in survival of patients with hepatocellular carcinoma between 1977 and 1996 in
        the United States. Hepatology 2001; 33: 62-65   PubMed

4      Kroll DJ, Shaw HS, Oberlies NH. Milk thistle nomenclature: why it matters in cancer research and pharmacokinetic
        studies. Integr Cancer Ther 2007; 6: 110-119   PubMed

5      Gazak R, Walterova D, Kren V. Silybin and silymarin--new and emerging applications in medicine. Curr Med Chem 2007;
        14: 315-338   PubMed

6      Singh RP, Agarwal R. A cancer chemopreventive agent silibinin, targets mitogenic and survival signaling in prostate
        cancer. Mutat Res 2004; 555: 21-32   PubMed

7      Jacobs BP, Dennehy C, Ramirez G, Sapp J, Lawrence VA. Milk thistle for the treatment of liver disease: a systematic
        review and meta-analysis. Am J Med 2002; 113: 506-515   PubMed

8      Lieber CS, Leo MA, Cao Q, Ren C, DeCarli LM. Silymarin retards the progression of alcohol-induced hepatic fibrosis in
        baboons. J Clin Gastroenterol 2003; 37: 336-339   PubMed

9      Singh RP, Sharma G, Dhanalakshmi S, Agarwal C, Agarwal R. Suppression of advanced human prostate tumor growth
        in athymic mice by silibinin feeding is associated with reduced cell proliferation, increased apoptosis, and inhibition of
        angiogenesis. Cancer Epidemiol Biomarkers Prev 2003; 12: 933-939   PubMed

10    Tyagi A, Agarwal C, Agarwal R. Inhibition of retinoblastoma protein (Rb) phosphorylation at serine sites and an increase
        in Rb-E2F complex formation by silibinin in androgen-dependent human prostate carcinoma LNCaP cells: role in prostate
        cancer prevention. Mol Cancer Ther 2002; 1: 525-532   PubMed

11    Singh RP, Dhanalakshmi S, Tyagi AK, Chan DC, Agarwal C, Agarwal R. Dietary feeding of silibinin inhibits advance
        human prostate carcinoma growth in athymic nude mice and increases plasma insulin-like growth factor-binding protein-
        3 levels. Cancer Res 2002; 62: 3063-3069   PubMed

12    Tyagi A, Bhatia N, Condon MS, Bosland MC, Agarwal C, Agarwal R. Antiproliferative and apoptotic effects of silibinin in
        rat prostate cancer cells. Prostate 2002; 53: 211-217   PubMed

13    Yang SH, Lin JK, Chen WS, Chiu JH. Anti-angiogenic effect of silymarin on colon cancer LoVo cell line. J Surg Res 2003;
        113: 133-138   PubMed

14    Agarwal C, Singh RP, Dhanalakshmi S, Tyagi AK, Tecklenburg M, Sclafani RA, Agarwal R. Silibinin upregulates the
        expression of cyclin-dependent kinase inhibitors and causes cell cycle arrest and apoptosis in human colon carcinoma
        HT-29 cells. Oncogene 2003; 22: 8271-8282   PubMed

15    Mohan S, Dhanalakshmi S, Mallikarjuna GU, Singh RP, Agarwal R. Silibinin modulates UVB-induced apoptosis via
        mitochondrial proteins, caspases activation, and mitogen-activated protein kinase signaling in human epidermoid
        carcinoma A431 cells. Biochem Biophys Res Commun 2004; 320: 183-189   PubMed

16    Mallikarjuna G, Dhanalakshmi S, Singh RP, Agarwal C, Agarwal R. Silibinin protects against photocarcinogenesis via
        modulation of cell cycle regulators, mitogen-activated protein kinases, and Akt signaling. Cancer Res 2004; 64: 6349-
        6356   PubMed

17    Singh RP, Tyagi AK, Zhao J, Agarwal R. Silymarin inhibits growth and causes regression of established skin tumors in
        SENCAR mice via modulation of mitogen-activated protein kinases and induction of apoptosis. Carcinogenesis 2002; 23:
        499-510   PubMed

18    Tyagi A, Agarwal C, Harrison G, Glode LM, Agarwal R. Silibinin causes cell cycle arrest and apoptosis in human bladder
        transitional cell carcinoma cells by regulating CDKI-CDK-cyclin cascade, and caspase 3 and PARP cleavages.
2004; 25: 1711-1720   PubMed

19    Tyagi AK, Agarwal C, Singh RP, Shroyer KR, Glode LM, Agarwal R. Silibinin down-regulates survivin protein and mRNA
        expression and causes caspases activation and apoptosis in human bladder transitional-cell papilloma RT4 cells.
        Biochem Biophys Res Commun
2003; 312: 1178-1184   PubMed

20    Chu SC, Chiou HL, Chen PN, Yang SF, Hsieh YS. Silibinin inhibits the invasion of human lung cancer cells via decreased
        productions of urokinase-plasminogen activator and matrix metalloproteinase-2. Mol Carcinog 2004; 40: 143-149


21    Lah J, Cui W, Hu KQ. Inhibitory effects and mechanisms of silibinin on growth of human hepatoma cell lines. Hepatology
        2005; 42: Suppl 309A (Abstract)  

22    Varghese L, Agarwal C, Tyagi A, Singh RP, Agarwal R. Silibinin efficacy against human hepatocellular carcinoma. Clin
        Cancer Res
2005; 11: 8441-8448   PubMed

23    Johnson PJ. The role of serum alpha-fetoprotein estimation in the diagnosis and management of hepatocellular
        carcinoma. Clin Liver Dis 2001; 5: 145-159   PubMed

24    Shirabe K, Takenaka K, Gion T, Shimada M, Fujiwara Y, Sugimachi K. Significance of alpha-fetoprotein levels for
        detection of early recurrence of hepatocellular carcinoma after hepatic resection. J Surg Oncol 1997; 64: 143-146


25    Peng SY, Chen WJ, Lai PL, Jeng YM, Sheu JC, Hsu HC. High alpha-fetoprotein level correlates with high stage, early
        recurrence and poor prognosis of hepatocellular carcinoma: significance of hepatitis virus infection, age, p53 and beta-
        catenin mutations. Int J Cancer 2004; 112: 44-50   PubMed

26    Osaki M, Oshimura M, Ito H. PI3K-Akt pathway: its functions and alterations in human cancer. Apoptosis 2004; 9: 667-
        676   PubMed

27    Lawlor MA, Alessi DR. PKB/Akt: a key mediator of cell proliferation, survival and insulin responses? J Cell Sci 2001;
        114: 2903-2910   PubMed

28    Yao R, Cooper GM. Requirement for phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve growth factor.
1995; 267: 2003-2006   PubMed

29    Li J, Yen C, Liaw D, Podsypanina K, Bose S, Wang SI, Puc J, Miliaresis C, Rodgers L, McCombie R, Bigner SH, Giovanella
        BC, Ittmann M, Tycko B, Hibshoosh H, Wigler MH, Parsons R. PTEN, a putative protein tyrosine phosphatase gene
        mutated in human brain, breast, and prostate cancer. Science 1997; 275: 1943-1947   PubMed

30    Steck PA, Pershouse MA, Jasser SA, Yung WK, Lin H, Ligon AH, Langford LA, Baumgard ML, Hattier T, Davis T, Frye C,
        Hu R, Swedlund B, Teng DH, Tavtigian SV. Identification of a candidate tumour suppressor gene, MMAC1, at
        chromosome 10q23.3 that is mutated in multiple advanced cancers. Nat Genet 1997; 15: 356-362   PubMed

31    Wan XW, Jiang M, Cao HF, He YQ, Liu SQ, Qiu XH, Wu MC, Wang HY. The alteration of PTEN tumor suppressor
        expression and its association with the histopathological features of human primary hepatocellular carcinoma. J Cancer
        Res Clin Oncol
2003; 129: 100-106   PubMed

32    Liu LZ, Zhou XD, Qian G, Shi X, Fang J, Jiang BH. AKT1 amplification regulates cisplatin resistance in human lung cancer
        cells through the mammalian target of rapamycin/p70S6K1 pathway. Cancer Res 2007; 67: 6325-6332   PubMed

33    Sansal I, Sellers WR. The biology and clinical relevance of the PTEN tumor suppressor pathway. J Clin Oncol 2004; 22:
        2954-2963   PubMed

34    Grunstein M. Histone acetylation in chromatin structure and transcription. Nature 1997; 389: 349-352   PubMed

35    Cress WD, Seto E. Histone deacetylases, transcriptional control, and cancer. J Cell Physiol 2000; 184: 1-16   PubMed

36    Song J, Noh JH, Lee JH, Eun JW, Ahn YM, Kim SY, Lee SH, Park WS, Yoo NJ, Lee JY, Nam SW. Increased expression of
        histone deacetylase 2 is found in human gastric cancer. APMIS 2005; 113: 264-268   PubMed

37    Hu KQ, Yu CH, Mineyama Y, McCracken JD, Hillebrand DJ, Hasan M. Inhibited proliferation of cyclooxygenase-2
        expressing human hepatoma cells by NS-398, a selective COX-2 inhibitor. Int J Oncol 2003; 22: 757-763   PubMed

38    Cui W, Yu CH, Hu KQ. In vitro and in vivo effects and mechanisms of celecoxib-induced growth inhibition of human
        hepatocellular carcinoma cells. Clin Cancer Res 2005; 11: 8213-8221   PubMed

39    Meuillet EJ, Mahadevan D, Berggren M, Coon A, Powis G. Thioredoxin-1 binds to the C2 domain of PTEN inhibiting
        PTEN's lipid phosphatase activity and membrane binding: a mechanism for the functional loss of PTEN's tumor
        suppressor activity. Arch Biochem Biophys 2004; 429: 123-133   PubMed

40    Hall PA, Levison DA, Woods AL, Yu CC, Kellock DB, Watkins JA, Barnes DM, Gillett CE, Camplejohn R, Dover R.
        Proliferating cell nuclear antigen (PCNA) immunolocalization in paraffin sections: an index of cell proliferation with
        evidence of deregulated expression in some neoplasms. J Pathol 1990; 162: 285-294   PubMed

41    Deshpande A, Sicinski P, Hinds PW. Cyclins and cdks in development and cancer: a perspective. Oncogene 2005; 24:
        2909-2915   PubMed

42    Seville LL, Shah N, Westwell AD, Chan WC. Modulation of pRB/E2F functions in the regulation of cell cycle and in cancer.
        Curr Cancer Drug Targets
2005; 5: 159-170   PubMed

43    Megargee EI, Cook PE. Negative response bias and the MMPI overcontrolled-hostility scale: a response to Deiker. J
        Consult Clin Psychol
1975; 43: 725-729   PubMed

44    Xu J, You C, Zhang S, Huang S, Cai B, Wu Z, Li H. Angio-genesis and cell proliferation in human craniopharyngioma
        xenografts in nude mice. J Neurosurg 2006; 105: 306-310   PubMed

45    Tutton MG, George ML, Eccles SA, Burton S, Swift RI, Abulafi AM. Use of plasma MMP-2 and MMP-9 levels as a
        surrogate for tumour expression in colorectal cancer patients. Int J Cancer 2003; 107: 541-550   PubMed

46    Mitsuuchi Y, Johnson SW, Selvakumaran M, Williams SJ, Hamilton TC, Testa JR. The phosphatidylinositol 3-kinase/AKT
        signal transduction pathway plays a critical role in the expression of p21WAF1/CIP1/SDI1 induced by cisplatin and
        paclitaxel. Cancer Res 2000; 60: 5390-6394   PubMed

47    Rahman MA, Kyriazanos ID, Ono T, Yamanoi A, Kohno H, Tsuchiya M, Nagasue N. Impact of PTEN expression on the
        outcome of hepatitis C virus-positive cirrhotic hepatocellular carcinoma patients: possible relationship with COX II and
        inducible nitric oxide synthase. Int J Cancer 2002; 100: 152-157   PubMed

48    Dong-Dong L, Xi-Ran Z, Xiang-Rong C. Expression and significance of new tumor suppressor gene PTEN in primary
        liver cancer. J Cell Mol Med 2003; 7: 67-71   PubMed

49    Hu TH, Huang CC, Lin PR, Chang HW, Ger LP, Lin YW, Changchien CS, Lee CM, Tai MH. Expression and prognostic role
        of tumor suppressor gene PTEN/MMAC1/TEP1 in hepatocellular carcinoma. Cancer 2003; 97: 1929-1940   PubMed

50    Ladas EJ, Kelly KM. Milk thistle: is there a role for its use as an adjunct therapy in patients with cancer? J Altern
        Complement Med
2003; 9: 411-416   PubMed

51    Sah NK, Khan Z, Khan GJ, Bisen PS. Structural, functional and therapeutic biology of survivin. Cancer Lett 2006; 244:
        164-171   PubMed

52    Kinoshita S, Hirai R, Yamano T, Yuasa I, Tsukuda K, Shimizu N. Angiogenesis inhibitor TNP-470 can suppress
        hepatocellular carcinoma growth without retarding liver regeneration after partial hepatectomy. Surg Today 2004; 34:
        40-46   PubMed

53    Besson A, Robbins SM, Yong VW. PTEN/MMAC1/TEP1 in signal transduction and tumorigenesis. Eur J Biochem 1999;
        263: 605-611   PubMed

54    Paramio JM, Navarro M, Segrelles C, Gomez-Casero E, Jorcano JL. PTEN tumour suppressor is linked to the cell cycle
        control through the retinoblastoma protein. Oncogene 1999; 18: 7462-7468   PubMed

55    Weng LP, Brown JL, Eng C. PTEN coordinates G(1) arrest by down-regulating cyclin D1 via its protein phosphatase
        activity and up-regulating p27 via its lipid phosphatase activity in a breast cancer model. Hum Mol Genet 2001; 10: 599-
        604   PubMed

56    Wu ZX, Song TB, Li DM, Zhang XT, Wu XL. Overexpression of PTEN suppresses growth and induces apoptosis by
        inhibiting the expression of survivin in bladder cancer cells. Tumour Biol 2007; 28: 9-15   PubMed


                  S- Editor  Liu Y    L- Editor  Alpini GD    E- Editor  Li HY




Reviews Add


Related Articles:
Recurrence or metastasis of HCC:predictors, early detection and experimental antiangiogenic therapy
Inhibitory effect of IGF- II antisense RNA on malignant phenotype of hepatocellular carcinoma
Analysis of in vivo patterns of caspase 3 gene expression in primary hepatocellular carcinoma and its relationship to p21WAF1 expression and hepatic apoptosis
Hepatocellular carcinoma in central Sydney:a 10-year review of patients seen in a medical oncology department
Characterization of six tumor suppressor genes and microsatellite instability in hepatocellular carcinoma in southern African blacks