Repression of the human papillomavirus E6 gene initiates p53-dependent, telomerase-independent senescence and apoptosis in HeLa cervical carcinoma cells

Cervical cancer cells express high-risk human papillomavirus (HPV) E6 and E7 proteins. When both HPV oncogenes are repressed in HeLa cervical carcinoma cells, the dormant p53 and retinoblastoma (Rb) tumor suppressor pathways are activated, and the cells undergo senescence in the absence of apoptosis. When the E6 gene is repressed in cells that continue to express an E7 gene, the p53 pathway, but not the Rb pathway, is activated, and both senescence and apoptosis are triggered. To determine the role of p53 signaling in senescence or apoptosis after repression of HPV oncogenes, we introduced a dominant-negative allele of p53 into HeLa cells. Dominant-negative p53 prevented senescence and apoptosis when E6 alone was repressed but did not inhibit senescence when both E6 and E7 were repressed. To determine whether reduced telomerase activity was involved in senescence or apoptosis after E6 repression, we generated HeLa cells stably expressing an exogenous hTERT gene, which encodes the catalytic subunit of telomerase. Although these cells contained markedly elevated telomerase activity and elongated telomeres, hTERT expression did not prevent senescence and apoptosis when E6 alone was repressed. These results demonstrate that when the Rb tumor suppressor pathway is inactivated by the E7 protein, E6 repression activates p53 signaling, which in turn is required for growth inhibition, senescence, and apoptosis. Thus, sustained inactivation of the p53 pathway by the E6 protein is required for maintenance of the proliferative phenotype of HeLa cervical carcinoma cells.     

Restoration of p53 tumor suppressor pathway in human cervical carcinoma cells by sodium arsenite

In most cervical cancer cells, p53 and Rb are disrupted by human papillomaviruses (HPVs) E6 and E7, respectively. Restoration of p53 or Rb function by blocking E6/p53 or E7/Rb pathway might be a potential therapeutic purpose for these cancer cells. Treatment with sodium arsenite (SA) resulted in significant repression of E6 and E7 mRNA levels in SiHa cells. After E6 and E7 repression, p53 was dramatically induced and accumulated in cellular nuclei and Rb was also induced. Two p53-responsive genes, p21(waf1/cip1) and mdm2, were induced after SA treatment. Furthermore, SA also reduced the expressions of Cdc25A and cyclin B, blocked cell cycle progression at G2/M phase, and induced apoptosis in SiHa cells. SA-induced apoptosis was greatly reduced by expression of a dominant-negative mutated p53. In this study, we have first demonstrated that SA did repress E6 and E7 oncogenes, restore the p53 tumor suppressor pathway and induce apoptosis in SiHa cells. Therefore, it would be a potential strategy to promote SA as therapeutic purpose for HPV-positive cancer cells.     

Endogenous human papillomavirus E6 and E7 proteins differentially regulate proliferation,senescence, and apoptosis in HeLa cervical carcinoma cells

Cervical cancer cells express high-risk human papillomavirus (HPV) E6 and E7 proteins, and repression of HPV gene expression causes the cells to cease proliferation and undergo senescence. However, it is not known whether both HPV proteins are required to maintain the proliferative state of cervical cancer cells, or whether mutations that accumulate during carcinogenesis eliminate the need for one or the other of them. To address these questions, we used the bovine papillomavirus E2 protein to repress the expression of either the E6 protein or the E7 protein encoded by integrated HPV18 DNA in HeLa cervical carcinoma cells. Repression of the E7 protein activated the Rb pathway but not the p53 pathway and triggered senescence, whereas repression of the E6 protein activated the p53 pathway but not the Rb pathway and triggered both senescence and apoptosis. Telomerase activity, cyclin-dependent kinase activity, and expression of c-myc were markedly inhibited by repression of either E6 or E7. These results demonstrate that continuous expression of both the E6 and the E7 protein is required for optimal proliferation of cervical carcinoma cells and that the two viral proteins exert distinct effects on cell survival and proliferation. Therefore, strategies that inhibit the expression or activity of either viral protein are likely to inhibit the growth of HPV-associated cancers.     

Human papillomaviruses type 16+ and 18+ cervical carcinoma cells are sensitive to staurosporine-mediated apoptosis

We have recently shown that staurosporine (ST) can trigger apoptosis of CaSki and HeLa cervical tumor cells from G2/M checkpoint, though the mechanism remains elusive. In this study, we reported that ST induced the inhibition of E6 and E7 viral oncogene and MDM2 expression, while it led to increased levels of p53, which was transiently located to mitochondria. Additionally, the proteins of the p53-regulated genes, p21(WAF1) and Bax, were increased with a similar time, while Bcl-2 and Bcl-X(L) expression was lowered. Upon ST treatment, the cytochrome c was released into the cytosol and, then, activation of caspases-9 and -3 led to Poly(ADP)RibosePolymerase (PARP) cleavage. Finally, characteristic morphological signs confirmed the apoptosis execution. Thus, taken together, all these observations suggest that apoptosis can be reactivated in HPV-positive human carcinoma cells and highlight that ST could be used as a potently chemotherapy agent to enhance the sensitivity of tumor cells to apoptosis.     

BF12, a Novel Benzofuran,Exhibits Antitumor Activity by Inhibiting Microtubules and the PI3K/Akt/mTOR Signaling Pathway in Human Cervical Cancer Cells

BF12 [(2E)‐3‐[6‐Methoxy‐2‐(3,4,5‐trimethoxybenzoyl)‐1‐benzofuran‐5‐yl]prop‐2‐enoic acid], a novel derivative of combretastatin A‐4 (CA‐4), was previously found to inhibit tumor cell lines, with a particularly strong inhibitory effect on cervical cancer cells. In this study, we investigated the microtubule polymerization effects and apoptosis signaling mechanism of BF12. BF12 showed a potent efficiency against cervical cancer cells, SiHa and HeLa, with IC₅₀ values of 1.10 and 1.06 μm , respectively. The cellular mechanism studies revealed that BF12 induced G2/M phase arrest and apoptosis in SiHa and HeLa cells, which were associated with alterations in the expression of the cell G2/M cycle checkpoint‐related proteins (cyclin B1 and cdc2) and alterations in the levels of apoptosis‐related proteins (P53, caspase‐3, Bcl‐2, and Bax) of these cells, respectively. Western blot analysis showed that BF12 inhibited the PI3 K/Akt/mTOR signaling pathway and induced apoptosis in human cervical cancer cells. BF12 was identified as a tubulin polymerization inhibitor, evidenced by the effective inhibition of tubulin polymerization and heavily disrupted microtubule networks in living SiHa and HeLa cells. By inhibiting the PI3 K/Akt/mTOR signaling pathway and inducing apoptosis in human cervical cancer cells, BF12 shows promise for use as a microtubule inhibitor.     

Cellular and molecular mechanisms of silibinin induces cell-cycle arrest and apoptosis on HeLa cells

Silibinin, an effective anti-cancer and chemopreventive agent in various epithelial cancer models, has been reported to inhibit cancer cell growth through mitogenic signalling pathways including cervical cancer. However, the underlying mechanisms are still not well elucidated. Here, we assessed the effect of silibinin on human cervical carcinoma cell cycle modulation, apoptosis induction and associated molecular alterations by employing HeLa cell line. Silibinin treatment of HeLa cells resulted in a G2 arrest and induced a decrease in cyclin-dependent kinases involved in both G1 and G2 progression. In addition, silibinin showed a dose-dependent and a time-dependent apoptotic death in HeLa cells in both the mitochondrial pathway and the death receptor-mediated pathway, providing a strong rationale for future studies evaluating preventive and/or intervention strategies for silibinin in cervical cancer pre-clinical models.     

Novel anthranilamide-pyrazolo[1,5-a]pyrimidine conjugates modulate the expression of p53-MYCN associated micro RNAs in neuroblastoma cells and cause cell cycle arrest and apoptosis

It has previously been shown that anthranilamide-pyrazolo[1,5-a]pyrimidine conjugates activate p53 and cause apoptosis in cervical cancer cells such as HeLa and SiHa. Here we establish the role of these conjugates in activating p53 pathway by phosphorylation at Ser15, 20 and 46 residues and downregulate key oncogenic proteins such as MYCN and Mdm2 in IMR-32 neuroblastoma cells. Compounds decreased the proliferation rate of neuroblastoma cells such as IMR-32, Neuro-2a, SK-N-SH. Compound treatment resulted in G2/M cell cycle arrest. The expression of p53 dependent genes such as p21, Bax, caspases was increased with concomitant decrease of the survival proteins as well as anti-apoptotic proteins such as Akt1, E2F1 and Bcl2. In addition the expression of important microRNAs such as miR-34a, c, miR-200b, miR-107, miR-542-5p and miR-605 were significantly increased that eventually lead to the activation of apoptotic pathway. Our data revealed that conjugates of this nature cause cell cycle arrest and apoptosis in IMR-32 cells [MYCN (+) with intact wild-type p53] by activating p53 signalling and provides a lead for the development of anti-cancer therapeutics.     

Growth suppression induced by downregulation of E6-AP expression in human papillomavirus-positive cancer cell lines depends on p53

The ubiquitin-protein ligase E6-AP is utilized by the E6 oncoprotein of human papillomaviruses (HPVs) associated with cervical cancer to target the tumor suppressor p53 for degradation. Here, we report that downregulation of E6-AP expression by RNA interference results in both the accumulation of p53 and growth suppression of the HPV-positive cervical cancer cell lines HeLa and SiHa. In addition, HeLa cells, in which p53 expression was suppressed by RNA interference, are significantly less sensitive to the downregulation of E6-AP expression with respect to growth suppression than parental HeLa cells. These data indicate that the anti-growth-suppressive properties of E6-AP in HPV-positive cells depend on its ability to induce p53 degradation.     

应用DNA芯片鉴定HPV18 E6-siRNA诱导HeLa 细胞凋亡的分子机制

高危型人乳头瘤病毒（human papillomavirus,HPV）的E6基因在宫颈癌的发生中起关键作用,特异siRNA能有效抑制宫颈癌HeLa 细胞内HPV18 E6基因的表达,诱导肿瘤细胞凋亡.为进一步探讨HPV18 E6-siRNA诱导HeLa 细胞凋亡的分子机制,针对HPV18-E6基因设计siRNA序列,利用人源U6启动子为模板,经PCR表达框架法体外扩增,转染宫颈癌HeLa细胞抑制HPV18 -E6基因表达,从而诱导肿瘤细胞凋亡.对转染前后HeLa细胞总RNA样品进行荧光标记后,与Agilent Human 1A寡核苷酸芯片杂交、扫描、数据分析及标准化处理,确定表达差异的基因并经荧光定量PCR对部分基因进行验证,结合PANTHER数据分析系统,将这些基因按照生物学功能进行归类,查阅GenBank数据库及相关文献,对其结果进行深入分析及讨论.在检测的18 716个基因和EST中,共筛出差异表达基因359个,其中307个基因表达上调,52个基因表达下调,主要包括细胞周期相关基因CCNG1、p21;凋亡相关基因CASP4、CASP6、IGFBP3、DFFA;泛素蛋白酶解途径相关基因E6-AP、UBE2C;角化细胞分化相关基因KRT4、KRT6E、KRT18;抑癌基因RECK、VHL等.研究结果表明,HPV18 -E6基因抑制引起的细胞凋亡效应主要是通过P53信号途径和泛素蛋白酶解信号途径调节细胞周期相关基因和凋亡相关基因的表达,从而抑制HeLa细胞增殖、促进细胞凋亡.同时,抑癌基因的激活,角化细胞分化和免疫相关基因的表达上调,都说明了E6抑制后肿瘤细胞恶性转化程度的下降.     

Spatiotemporal activation of caspase‐dependent and ‐independent pathways in staurosporine‐induced apoptosis of p53wt and p53mt human cervical carcinoma cells

Background information. Caspase‐dependent and ‐independent death mechanisms are involved in apoptosis in a variety of human carcinoma cells treated with antineoplastic compounds. Our laboratory has reported that p53 is a key contributor of mitochondrial apoptosis in cervical carcinoma cells after staurosporine exposure. However, higher mitochondrial membrane potential dissipation and greater DNA fragmentation were observed in p53^wt (wild‐type p53) HeLa cells compared with p53^mt (mutated p53) C‐33A cells. Here, we have studied events linked to the mitochondrial apoptotic pathway. Results. Staurosporine can induce death of HeLa cells via a cytochrome c/caspase‐9/caspase‐3 mitochondrial‐dependent apoptotic pathway and via a delayed caspase‐independent pathway. In contrast with p53^wt cells, p53^mt C‐33A cells exhibit firstly caspase‐8 activation leading to caspase‐3 activation and Bid cleavage followed by cytochrome c release. Attenuation of PARP‐1 [poly(ADP‐ribose) polymerase‐1] cleavage as well as oligonucleosomal DNA fragmentation in the presence of z‐VAD‐fmk points toward a major involvement of a caspase‐dependent pathway in staurosporine‐induced apoptosis in p53^wt HeLa cells, which is not the case in p53^mt C‐33A cells. Meanwhile, the use of 3‐aminobenzamide, a PARP‐1 inhibitor known to prevent AIF (apoptosis‐inducing factor) release, significantly decreases staurosporine‐induced death in these p53^mt carcinoma cells, suggesting a preferential implication of caspase‐independent apoptosis. On the other hand, we show that p53, whose activity is modulated by pifithrin‐α, isolated as a suppressor of p53‐mediated transactivation, or by PRIMA‐1 (p53 reactivation and induction of massive apoptosis), that reactivates mutant p53, causes cytochrome c release as well as mitochondrio—nuclear AIF translocation in staurosporine‐induced apoptosis of cervical carcinoma cells. Conclusions. The present paper highlights that staurosporine engages the intrinsic mitochondrial apoptotic pathway via caspase‐8 or caspase‐9 signalling cascades and via caspase‐independent cell death, as well as through p53 activity.     

Neuroprotection by lomerizine, a prophylactic drug for migraine, against hydrogen peroxide-induced hippocampal neurotoxicity

Cervical cancer is the most common cancer in Indian females and is associated with infection with high-risk Human papilloma viruses (HPVs) which encode viral oncoprotein E6 and E7. Estradiol has been established as a risk factor for cervical cancer and has been shown to play a synergistic role with viral oncoproteins. Curcumin (Diferuloyl methane), a chemopreventive agent, is a natural compound extracted from Curcuma longa that allows suppression and retardation of carcinogenesis in many types of cancer and is currently being tested in various human clinical trials as it has been found to be well tolerated at higher doses with a relatively well established safety profile. The objective of this study was to test the effect of curcumin on HPV-positive and negative cervical cancer cell lines HeLa, SiHa, CaSki, and C33A pretreated with estradiol. It was found that HPV-positive cells pretreated with estradiol show reduced apoptosis as compared to curcumin by itself. However, curcumin was able to counteract the proliferative response of estradiol, and induce apoptosis. There was no difference in percentage apoptosis as compared to estradiol pretreatment in HPV-negative cell line C33A. Molecular studies showed elevation of Telomerase, viral oncoproteins E6 and E7, PCNA, p16, Cyclin D1 in HPV-positive cell lines on treatment with estradiol but after treatment with curcumin the level of E7, PCNA, and Cyclin D1 was reduced but the level of E6, Telomerase, and p16 was unaltered. Furthermore, estradiol-pretreated HPV-negative cell line C33A showed reduction in level of Telomerase, PCNA, p16, and activation of both p53 and p73 tumor suppressor proteins, thus, demonstrating the importance of E6 in estradiol-mediated protective effect.