Survivin depletion inhibits tumor growth and enhances chemosensitivity in hepatocellular carcinoma

Survivin is a member of the inhibitor of apoptosis family, which has been suggested to be crucial in the control of cell division and inhibition of apoptosis. Expression of this protein has been observed in transformed cell lines and human tumor tissues, including those from colorectal cancer, but not in terminally differentiated adult tissues. Survivin mRNA expression has frequently been detected in hepatocellular carcinoma (HCC) and its protein expression has been demonstrated to be highly correlated with proliferation index rather than apoptotic index. The present study aimed to analyze the effect of survivin on the tumorigenicity and chemosensitivity of HCC via the establishment of an HCC cell line (PLC/PRF/5) with the stable knockdown of the survivin gene (PLC-k3). This cell line displayed significantly lower rates of survival and proliferation in assays of cell viability and proliferation, respectively, compared with those of the control cell line (PLC-v). In addition, PLC-k3 cells were more sensitive to cisplatin treatment, resulting in S phase arrest. These findings were further confirmed by an in vivo experiment. The data of the present study suggest that survivin is critical in promoting cell proliferation but not in inhibition of apoptosis, and enhances the chemosensitivity of HCC. Thus, the suppression of survivin expression in combination with cisplatin may contribute to the development of more effective treatments for HCC.published_or_final_versio

Knockdown of survivin gene suppresses the tumorigenicity and enhances chemosensitivity in hepatocellular carcinoma (HCC)

Survivin, the inhibitor of apoptosis (IAP) family member, was suggested to control cell division and inhibit apoptosis. Expression of this protein was only found in transformed cell lines and human tumor tissues, such as colorectal cancer, but not in terminally differentiated adult tissue. Survivin mRNA was frequently expressed in HCC and its protein expression was shown to be highly correlated to the proliferation index (Ki-67). The present study aimed at studying the effect of survivin on the tumorigenicity and chemosensitivity in HCC via the establishment of an HCC cell line (PLC) with the stably knockdown of survivin gene (PLC-k). The viability of PLC-k showed 13.3% and 33.3% lower than that of the cell line transfected with vector control (PLC-v) at 24- and 48-hour time points, respectively. Addition of cisplatin reduced the cell viability of PLC-k cells to 50% and 53% at 24- and 48-hour time points, respectively. Whereas PLC-v cells showed no significant change and 21% decrease in cell viability at 24- and 48-hour time points, respectively. To elucidate the cellular activity and the responses to chemotherapeutic drug treatment in PLC-k and PLC-v cells, apoptotic and proliferation assays were performed. By Annexin V staining, early apoptosis in the two cell lines were similar at 24 hour after cell seeding (PLC-v: 7.15% and PLC-k: 8.67%). After cisplatin treatment, the changes in the percentage of apoptotic cells were similar in the two cell lines, with the increase of around 1.4-fold and 2.2-fold at 24 and 48 hour time point, respectively. In the proliferation analysis, cells were seeded and cell numbers were counted at 24, 48 and 72 hour time points. Numbers of PLC-k cells were significantly lower than PLC-v cells in all the 3 time points, implying the proliferation rate of PLC-k was significantly lower than PLC-v. After 24-hour cisplatin treatment on the two cell lines, PLC-k showed significant decrease (40.4%) in cell number comparing to untreated cells, while there was no significant decrease in PLC-v cells. At 48-hour time point, more dramatic decrease in cell number was detected in PLC-k (63%) comparing to PLC-v (31.4%). In in vivo study, the cell lines were subcutaneously injected into nude mice for tumorigenicity assay. PLC-v induced tumor growth in all 12 mice, whereas PLC-k can only induce tumor growth in 2 out of 12 mice. Besides, the PLC-k-induced tumors grew dramatically slower than PLC-v-induced tumors. The above results demonstrated the role of survivin on tumor growth and chemosensitivity in HCC. The significant decrease in the cell viability of PLC-k and its responses to drug treatment may be due to the inhibition of cell proliferation rather than induction of apoptosis. To further investigate the function of survivin genes on cell proliferation and chemosensitivity in HCC, cell cycle analysis by flow cytometry and studying cell cycle protein expressions will be performed

Blockade of VEGF/VEGFR pathway enhances chemo-sensitivity in hepatocellular carcinoma

Background: Systemic and local chemotherapy for hepatocellular carcinoma (HCC) remains a tough task due to chemo-resistance of tumor cells to cytotoxic agents. Interaction between vascular endothelial growth factor (VEGF) and its receptors plays an important role in the proliferation and survival of tumor cells. Since HCC cells express high levels of VEGF and VEGFRs, we postulate that VEGF/VEGFR pathway might be involved in chemo-resistance of tumor cells to cytotoxic agents. Methods: In vitro, HCC cell lines with wild type (wt) p53 (HepG2), mutant/null p53 (PLC/Hep3B) and p53 stable transfectant (Hep3B/p53+) were treated with cisplatin, VEGF, PTK787 (a VEGFR inhibitor), and cisplatin combined with PTK787, respectively. Cell apoptosis was determined by Annexin V/PI staining, and the mechanism of cell death was explored by poly(ADP-ribose) polymerase (PARP) degradation, and alterations in pro-apoptotic and anti-apoptotic molecules. In vivo, the effects of PTK787 combined with cisplatin was evaluated in a HCC xenograft model in nude mice. Results: The three HCC cell lines express different levels of VEGFRs, Flt-1 and Flk-1. PTK787 enhanced cisplatin-induced PARP degradation and apoptosis in all the three cell lines tested, especially in PLC cells. PTK787, or cisplatin alone down-regulated the expression of pro-survival molecules, cyclin D1 and Bcl-2, and a synergistic effect was observed when PTK787 was combined with cisplatin. Administration of VEGF recombinant protein could abrogate the synergistic effects of PTK787 and cisplatin. VEGF treatment stimulated up-regulation of survivin in tumor cells, while knockdown of survivin reversed the protective effects of VEGF on cisplatin-induced cell apoptosis. Prominent retardation of tumor growth was observed in the HCC xenografts treated with PTK787 and cisplatin in vivo. Conclusions: This study demonstrated that VEGF/VEGFR pathway played an important role in protecting tumor cells from cisplatin-induced cell death, and blockade of VEGF/VEGFR pathway could enhance cisplatin-induced chemo-cytotoxicity in HCC

Identification of a novel 12p13.3 amplicon in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a distinct type of head and neck cancer commonly occurring in southern China. To decipher the molecular basis of this cancer, we performed high-resolution array CGH analysis on eight tumour lines and 10 primary tumours to identify the genes involved in NPC tumorigenesis. In this study, multiple regions of gain were consistently found at 1q21-q24, 7q11-12, 7q21-22., 11q13, 12p13, 12q13, 19p13 and 19q13. Importantly, a 2.1 Mb region at 12p13.31 was highly amplified in a NPC xenograft, xeno-2117. By FISH mapping, we have further delineated the amplicon to a 1.24 region flanked by RP11-319E16 and RP11-433J6. Copy number gains of this amplicon were confirmed in 21/41 (51%) primary tumours, while three cases (7.3%) showed high copy number amplification. Among the 13 genes within this amplicon, three candidate genes, lymphotoxin beta receptor (LTβR), tumour necrosis factor receptor superfamily memeber 1A (TNFRSF1R) and FLJ10665, were specifically over-expressed in the NPC xenograft with 12p13.3 amplification. However, only LTβR was frequently over-expressed in primary tumours. LTβR is a member of the TNF family of receptors, which can modulate NF-κB signalling pathways. Over-expression of LTβR in nasopharyngeal epithelial cells resulted in an increase of NF-κB activity and cell proliferation. In vivo study showed that suppression of LTβR by siRNA led to growth inhibition in the NPC tumour with 12p13.3 amplification. These findings implied that LTβR is a potential NPC-associated oncogene within the 12p13.3 amplicon and that its alteration is important in NPC tumorigenesis. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.link_to_subscribed_fulltex

Array-based comparative genomic hybridization analysis identified cyclin D1 as a target oncogene at 11q13.3 in nasopharyngeal carcinoma

Nasopharyegeal carcinoma is highly prevalent in Southern China and Southeast Asia. To unveil the molecular basis of this endemic disease, high-resolution comparative genomic hybridization arrays were used for systematic investigation of genomic abnormalities in 26 nasopharyngeal carcinoma samples. A comprehensive picture of genetic lesions associated with tumorigenesis of nasopharyngeal carcinoma was generated. Consistent chromosomal gains were frequently found on 1q, 3q, 8q, 11q, 12p, and 12q. High incidences of nonrandom losses were identified on chromosomes 3p, 9p, 11q, 14q, and 16q. In addition to previously characterized regions, we have identified several novel minimal regions of gains, including 3q27.3-28, 8q21-24, 11q13.1-13.3, and 12q13, which may harbor candidate nasopharyngeal carcinoma-associated oncogenes. In this study, gain of 11q13.1-13.3 was the most frequently detected chromosomal aberration and a 5.3-Mb amplicon was delineated at this region. Within this 11q13 amplicon, concordant amplification and overexpression of cyclin D1 (CCND1) oncogene was found in nasopharyngeal carcinoma cell lines, xenografts, and primary tumors. Knockdown of cyclin D1 by small interfering RNA in nasopharyngeal carcinoma cell lines led to significant decrease of cell proliferation. The findings suggest that cyclin D1 is a target oncogene at 11q13 in nasopharyngeal carcinoma and its activation plays a significant role in nasopharyngeal carcinoma tumorigenesis. ©2005 American Association for Cancer Research.link_to_OA_fulltex