HPV-positive Cervical Cancer Cells Research Articles

Ubiquitination of the HPV Oncoprotein E6 Is Critical for E6/E6AP-Mediated p53 Degradation.

High-risk Human papillomaviruses (HPVs) types are associated with more than 90% of premalignant and malignant squamous lesions of the uterine cervix. The E6 oncoprotein of high-risk HPVs is a key determinant in cell transformation because it induces the degradation of the host pro-apoptotic tumor suppressor p53. E6 recruits the intracellular ubiquitin ligase E6AP and subsequently induces proteasome-dependent p53 degradation. Neither E6 nor E6AP alone interact with p53; however, the precise mechanism of the functional regulation of the E6/E6AP/p53 complex is unclear. Here, we showed that the high-risk HPV E6 proteins are ubiquitinated during E6/E6AP/p53 complex assembly and degraded by the proteasome system. Increasing p53 expression enhanced E6/E6AP/p53 assembly and facilitated E6 ubiquitination and degradation. The dominant negative mutant of p53 R175H, which does not efficiently bind E6, decreased E6 ubiquitination and increased stability. Furthermore, we showed that the ubiquitin ligase E6AP is essential for E6 ubiquitination, and downregulation of E6AP expression increased E6 stability. We also showed that p53 R175H inhibited E6-mediated p53 degradation. Consistently, the host deubiquitinating enzyme USP15 removed ubiquitin chains from E6 proteins and inhibited E6-mediated p53 degradation. Crucially, ectopic expression of either p53 R175H or USP15 promoted p53-triggered apoptosis in human cervical cancer cells. Considering the importance of ubiquitinated E6 on p53 degradation, the disruption of E6 ubiquitination represents an attractive pharmacological intervention against HPV-positive human cervical cancer.ImportanceVirtually 100% of cervical cancers are linked to HPV infection. Commercial HPV vaccines are estimated to prevent up to 90% of HPV-associated cancers, while they do not eliminate persistent HPV infections and have no effect on the progression to malignancy. Hence, the development of novel therapeutic interventions against HPV is urgently required. The HPV oncoprotein E6 binds to the intracellular E3 ubiquitin ligase E6AP and p53 resulting in the degradation of p53. In this study, we demonstrate that HPV E6 is ubiquitinated by E6AP in presence of p53. Crucially, ubiquitination of E6 is important for p53 degradation and blockage of E6 ubiquitination negatively interferes with E6-mediated p53 degradation and enhances the apoptotic effects of p53 and the cytotoxicity of DNA damage in HPV-positive cervical cancer cells. Importantly, our data suggest that the HPV oncogene E6 might be an optimal pharmacologic.

Effects of the antifungal agent ciclopirox in HPV-positive cancer cells: Repression of viral E6/E7 oncogene expression and induction of senescence and apoptosis.

The malignant growth of human papillomavirus (HPV)-positive cancer cells is dependent on the continuous expression of the viral E6/E7 oncogenes. Here, we examined the effects of iron deprivation on the phenotype of HPV-positive cervical cancer cells. We found that iron chelators, such as the topical antifungal agent ciclopirox (CPX), strongly repress HPV E6/E7 oncogene expression, both at the transcript and protein level. CPX efficiently blocks the proliferation of HPV-positive cancer cells by inducing cellular senescence. Although active mTOR signaling is considered to be critical for the cellular senescence response towards a variety of prosenescent agents, CPX-induced senescence occurs under conditions of severely impaired mTOR signaling. Prolonged CPX treatment leads to p53-independent Caspase-3/7 activation and induction of apoptosis. CPX also eliminates HPV-positive cancer cells under hypoxic conditions through induction of apoptosis. Taken together, these results show that iron deprivation exerts profound antiviral and antiproliferative effects in HPV-positive cancer cells and suggest that iron chelators, such as CPX, possess therapeutic potential as HPV-inhibitory, prosenescent and proapoptotic agents in both normoxic and hypoxic environments.

Knockdown of METTL14 inhibits the growth and invasion of cervical cancer

Background: Increasing evidence has revealed that N6-methyladenosine (m6A) modification is implicated in multiple biological functions in mammals. Methyltransferase-like 14 (METTL14), an important component of m6A modification, has been reported to play important roles in the pathogenesis of acute myeloid leukaemia and hepatocellular carcinoma metastasis. However, its role in cervical cancer remains unclear. Methods: Expression of METTL14 was knocked down by shRNA-METTL14 interference in HPV-positive and HPV-negative cervical cancer cell lines SiHa and C33a. CCK8, colony formation, wound-healing, and Transwell assays were performed to evaluate the effects of METTL14 knockdown on the proliferation, migration and invasion abilities of SiHa and C33a cells. Flow cytometry analysis was utilized to detect cell cycle distribution, and the expression of related proteins was examined by western blot analysis. Results: Bioinformatics analysis demonstrated that up-regulation of METTL14 acted as an adverse prognostic factor for overall survival in cervical cancer patients. We demonstrated that down-regulation of METTL14 inhibited the proliferation, migration and invasion abilities of SiHa and C33a cells. Moreover, silencing METTL14 induced cell cycle arrest in cervical cancer cells. METTL14 knockdown suppressed the PI3K/Akt/mTOR signaling pathway by decreasing the phosphorylation of Akt and mTOR, and the expression of downstream apoptosis-related proteins was also impacted. Conclusions: In conclusion, these data suggest an important oncogenic role of METTL14 in the growth and invasion of both HPV-positive and HPV-negative cervical cancer cells.

Knockdown of METTL14 inhibits the growth and invasion of cervical cancer.

BackgroundIncreasing evidence has revealed that N6-methyladenosine (m6A) modification is implicated in multiple biological functions in mammals. Methyltransferase-like 14 (METTL14), an important component of m6A modification, has been reported to play important roles in the pathogenesis of acute myeloid leukaemia and hepatocellular carcinoma metastasis. However, its role in cervical cancer remains unclear.MethodsExpression of METTL14 was knocked down by shRNA-METTL14 interference in HPV-positive and HPV-negative cervical cancer cell lines SiHa and C33a. CCK8, colony formation, wound-healing, and Transwell assays were performed to evaluate the effects of METTL14 knockdown on the proliferation, migration and invasion abilities of SiHa and C33a cells. Flow cytometry analysis was utilized to detect cell cycle distribution, and the expression of related proteins was examined by western blot analysis.ResultsBioinformatics analysis demonstrated that up-regulation of METTL14 acted as an adverse prognostic factor for overall survival in cervical cancer patients. We demonstrated that down-regulation of METTL14 inhibited the proliferation, migration and invasion abilities of SiHa and C33a cells. Moreover, silencing METTL14 induced cell cycle arrest in cervical cancer cells. METTL14 knockdown suppressed the PI3K/Akt/mTOR signaling pathway by decreasing the phosphorylation of Akt and mTOR, and the expression of downstream apoptosis-related proteins was also impacted.ConclusionsIn conclusion, these data suggest an important oncogenic role of METTL14 in the growth and invasion of both HPV-positive and HPV-negative cervical cancer cells.

Benzothiazole derivative bearing amide moiety induces p53-mediated apoptosis in HPV16 positive cervical cancer cells.

In our previous study, we screened the anti-cancer properties of 10 benzothiazole derivatives in cervical cancer cell lines. In the present study, we aimed to delineate the mechanism of the apoptotic pathway (whether intrinsic or extrinsic) following the treatment of N-(4-(benzo[d]thiazol-2-yl)phenyl)-5-chloro-2-methoxybenzamide (named as A-07) on cervical cancer cell lines. Cellular stress by reactive oxygen species was measured using DCFDA dye by flowcytometry. Protein expression and localization was checked by immunofluorescence for γH2A.X, TP53, and CASP-3. Expression profiles of BAX and BCL-2 was done by semi-quantitative RT-PCR and PARP-1 (Poly(ADP-ribose) polymerase-1) by Western blot analysis. Bioinformatic studies were done using PDB websites, metaPocket 2.0 server, YASARA software and Discovery Studio 3.5 Visualizer. We demonstrate that the compound A-07 leads to ROS generation and double strand breaks in SiHa and C-33A cells. The induction of apoptosis in SiHa cells is associated with increased nuclear expression of the tumor suppressor protein, TP53. The shift in BAX/BCL-2 ratio, increased expression of Caspase-3 and cleaved Poly(ADP-ribose) polymerase-1 favour apoptotic signal in SiHa. In silico studies revealed that A-07 has inhibiting capabilities to the E6/E6AP/P53 complex. Our data suggest that treatment of A-07 causes p53 and caspase dependent apoptosis in HPV 16 infected SiHa cells.

Targeted juglone blocks the invasion and metastasis of HPV-positive cervical cancer cells

Human papillomaviruses (HPVs), for instance, HPV 16 and HPV 18, are concerned associated with cervical cancer. Thus, it is essential to suppress HPVs-in HPV-positive cervical cancer for treating cervical cancer. The purpose of this study was to explore the proposed molecular mechanisms, which that underlies the antintumor potential of juglone to treat of HPV-positive on cervical cancer cells. The results showed that juglone suppressed HPV-positive cell growth in a dose- and time-dependent way. In addition, cell invasion and metastasis were also inhibited by juglone. Nevertheless, when pin 1 was knocked down in HPV-positive cells, cell proliferation, invasion and metastasis were reduced. This study was designed to acquire an understanding of the mechanism of invasion and metastasis in HPV-positive cells suppressed by juglone. It provides evidence of the advantageous use of juglone in the future.

Puerarin suppresses cell growth and migration in HPV-positive cervical cancer cells by inhibiting the PI3K/mTOR signaling pathway.

Puerarin is an effective component that is present in high concentrations in the Pueraria lobata plant and is extensively distributed throughout nature. Puerarin possesses a number of pharmacological effects and has strong pharmacological activity with few side effects and extensive clinical applications. The aim of the present study was to explore the effects of Puerarin on the apoptosis of human papillomavirus (HPV)-positive cervical cancer cells and the underlying molecular mechanisms. MTT assay, lactate dehydrogenase activity and Annexin V/fluorescein isothiocyanate/propidium iodide analysis were used to analyze cell growth of HPV-positive HeLa cervical cancer cells treated with Puerarin. Western blotting was performed to measure protein expression in the treated cells. Puerarin significantly reduced cell proliferation and induced apoptosis in HeLa cells. In addition, it was observed that Puerarin significantly enhanced caspase-3/9 activities and significantly increased B-cell lymphoma 2-asscoiate X protein expression in HeLa cells. Puerarin suppressed phosphatidylinositol-3 kinase (PI3K), phosphorylated (p)-protein kinase B (Akt) and p-mammalian target of rapamycin (mTOR) protein expression in HeLa cells. These results indicate that Puerarin induces apoptosis in HPV-positive HeLa cervical cancer cells via inhibiting PI3K/Akt/mTOR signaling.

PI3K/AKT/mTOR Signaling Regulates the Virus/Host Cell Crosstalk in HPV-Positive Cervical Cancer Cells.

Human papillomavirus (HPV)-induced cancers will remain a significant clinical challenge for decades. Thus, the development of novel treatment strategies is urgently required, which should benefit from improving our understanding of the mechanisms of HPV-induced cell transformation. This should also include analyses of hypoxic tumor cells, which represent a major problem for cancer therapy. Recent evidence indicates that the PI3K/AKT/mTOR network plays a key role for the virus/host cell crosstalk in both normoxic and hypoxic HPV-positive cancer cells. In normoxic cells, the efficacy of the senescence induction upon experimental E6/E7 repression depends on active mTORC1 signaling. Under hypoxia, however, HPV-positive cancer cells can evade senescence due to hypoxic impairment of mTORC1 signaling, albeit the cells strongly downregulate E6/E7. Hypoxic repression of E6/E7 is mediated by the AKT kinase, which is activated under hypoxia by its canonical upstream regulators mTORC2 and PI3K. This review highlights our current knowledge about the oxygen-dependent crosstalk of the PI3K/AKT/mTOR signaling circuit with the HPV oncogenes and the phenotypic state of the host cell. Moreover, since the PI3K/AKT/mTOR pathway is considered to be a promising target for anticancer therapy, we discuss clinical implications for the treatment of HPV-positive cervical and head and neck squamous cell carcinomas.

Optimisation of Folate-Mediated Liposomal Encapsulated Arsenic Trioxide for Treating HPV-Positive Cervical Cancer Cells In Vitro.

High-risk human papilloma virus (HPV) infection is directly associated with cervical cancer development. Arsenic trioxide (ATO), despite inducing apoptosis in HPV-infected cervical cancer cells in vitro, has been compromised by toxicity and poor pharmacokinetics in clinical trials. Therefore, to improve ATO’s therapeutic profile for HPV-related cancers, this study aims to explore the effects of length of ligand spacers of folate-targeted liposomes on the efficiency of ATO delivery to HPV-infected cells. Fluorescent ATO encapsulated liposomes with folic acid (FA) conjugated to two different PEG lengths (2000 Da and 5000 Da) were synthesised, and their cellular uptake was examined for HPV-positive HeLa and KB and HPV-negative HT-3 cells using confocal microscopy, flow cytometry, and spectrophotometer readings. Cellular arsenic quantification and anti-tumour efficacy was evaluated through inductively coupled plasma-mass spectrometry (ICP-MS) and cytotoxicity studies, respectively. Results showed that liposomes with a longer folic acid-polyethylene glycol (FA-PEG) spacer (5000 Da) displayed a higher efficiency in targeting folate receptor (FR) + HPV-infected cells without increasing any inherent cytotoxicity. Targeted liposomally delivered ATO also displayed superior selectivity and efficiency in inducing higher cell apoptosis in HPV-positive cells per unit of arsenic taken up than free ATO, in contrast to HT-3. These findings may hold promise in improving the management of HPV-associated cancers.

Innervation of cervical carcinoma is mediated by cancer-derived exosomes

ObjectiveRecently, our laboratory identified sensory innervation within head and neck squamous cell carcinomas (HNSCCs) and subsequently defined a mechanism whereby HNSCCs promote their own innervation via the release of exosomes that stimulate neurite outgrowth. Interestingly, we noted that exosomes from human papillomavirus (HPV)-positive cell lines were more effective at promoting neurite outgrowth than those from HPV-negative cell lines. As nearly all cervical tumors are HPV-positive, we hypothesized that these findings would extend to cervical cancer. MethodsWe use an in vitro assay with PC12 cells to quantify the axonogenic potential of cervical cancer exosomes. PC12 cells are treated with cancer-derived exosomes, stained with the pan-neuronal marker (β-III tubulin) and the number of neurites quantified. To assess innervation in cervical cancer, we immunohistochemically stained cervical cancer patient samples for β-III tubulin and TRPV1 (sensory marker) and compared the staining to normal cervix. ResultsHere, we show the presence of sensory nerves within human cervical tumors. Additionally, we show that exosomes derived from HPV-positive cervical cancer cell lines effectively stimulate neurite outgrowth. ConclusionsThese data identify sensory nerves as components of the cervical cancer microenvironment and suggest that tumor- derived exosomes promote their recruitment.

Effect of adenovirus delivered tissue inhibitor of metalloproteinases 3 on the irradiation sensitivity of human papillomavirus positive cervical cancer cells

To explore the effects of adenovirus-delivered tissue inhibitor of metalloprotein- ases-3 (Ad-TIMP-3) on the irradiation sensitivity of human papillomavirus (HPV)-positive cervical cancer cells. An adenovirus expressing TIMP-3 (Ad-TIMP-3), alone or in combination with irradiation,was used to treat HPV-positive cervical cancer cells HeLa-Luc and CaSki. The effects of Ad-TIMP-3 on the proliferation of HeLa-Luc and CaSki cells were detected with MTT assay. The effect of the combination of Ad-TIMP-3 and X-ray on the proliferation of cells were determined by clone formation assay. Twenty nude mice were equally randomly divided into four groups: normal control group,Ad-TIMP-3 group,X-ray group,and combination group. The size of tumor was measured separately,and tumor growth curves were drawn. Ad-TIMP-3 significantly inhibited the proliferation of HPV-positive cervical cancer cells in a dose-dependent manner. Combination of Ad-TIMP-3 and X-ray significantly decreased the clones of HeLa-Luc and CaSki than Ad-TIMP-3 or X-ray alone (P<0.05). The tumor weights were (0.216±0.098), (0.276±0.073), and (0.044±0.043) g, respectively, in Ad-TIMP-3 group, X-ray group,and combination group, which were all significantly lower than that in normal control group [(0.534±0.218) g] (all P<0.05). In addition,the tumor weight in the combination group was significantly lower than that in Ad-TIMP-3 group and X-ray group (both P<0.05). The tumor inhibition rate was 59.60%, 48.30%, and 91.80% in X-ray group, Ad-TIMP-3 group and combination group, respectively. Ad-TIMP-3 can effectively inhibit the proliferation of cervical cancer cells. When combined with X-ray,it can remarkably increase the irradiation sensitivity of HPV-positive cervical cancer cells,and thus suppress the tumorigenesis capability of these cells in vivo.

Long non-coding RNA FAM83H-AS1 is regulated by human papillomavirus 16 E6 independently of p53 in cervical cancer cells

High-risk human papillomavirus (HPV) infection is one of the first events in the process of carcinogenesis in cervical and head and neck cancers. The expression of the viral oncoproteins E6 and E7 are essential in this process by inactivating the tumor suppressor proteins p53 and Rb, respectively, in addition to their interactions with other host proteins. Non-coding RNAs, such as long non-coding RNAs (lncRNAs) have been found to be dysregulated in several cancers, suggesting an important role in tumorigenesis. In order to identify host lncRNAs affected by HPV infection, we expressed the high-risk HPV-16 E6 oncoprotein in primary human keratinocytes and measured the global lncRNA expression profile by high-throughput sequencing (RNA-seq). We found several host lncRNAs differentially expressed by E6 including GAS5, H19, and FAM83H-AS1. Interestingly, FAM83H-AS1 was found overexpressed in HPV-16 positive cervical cancer cell lines in an HPV-16 E6-dependent manner but independently of p53 regulation. Furthermore, FAM83H-AS1 was found to be regulated through the E6-p300 pathway. Knockdown of FAM83H-AS1 by siRNAs decreased cellular proliferation, migration and increased apoptosis. FAM83H-AS1 was also found to be altered in human cervical cancer tissues and high expression of this lncRNA was associated with worse overall survival, suggesting an important role in cervical carcinogenesis.

The expression and significance of lncRNA HOST2 and microRNA let-7b in HPV-positive cervical cancer tissues and cell lines.

This study attempted to investigate the expression and significance of lncRNA HOST2 (human ovarian cancer-specific transcript 2) and microRNA let-7b in human papillomavirus (HPV)-positive cervical cancer (CC) tissues and cell lines. The expression of levels of HOST2 and let-7b were detected by qRT-PCR in HPV-positive CC tissues and cell lines. The HPV-positive CaSki and HeLa cells were divided into the Blank, NC, pcDNA3.0-HOST2, siHOST2, let-7b mimic, and pcDNA3.0-HOST2+let-7b mimic groups. Dual-luciferase reporter gene assay was employed to verify the targeting relationship between HOST2 and let-7b, MTT and flow cytometry to determinate cell proliferation and apoptosis, and wound-healing and transwell assays to evaluate cell migration and invasion capabilities. HOST2 was up-regulated but let-7b was down-regulated in HPV-positive CC tissues and cells. Dual-luciferase reporter gene assay confirmed the targeting relationship between HOST2 and let-7b. Over-expressed HOST2 reduced let-7b expression, promoted proliferation migration and invasion and inhibited the apoptosis of CaSki and HeLa cells; however, silencing HOST2 or overexpressing let-7b enhanced the expression of let-7b, inhibited proliferation migration and invasion, and promoted the apoptosis of CaSki and HeLa cells, and let-7b mimic could reverse the promoting effect of HOST2 on the growth of CC cells. HOST2 was upregulated in HPV-positive CC tissues and cells, which could promote the proliferation, migration and invasion, but inhibit the apoptosis of HPV-positive CC cells via inhibition of let-7b.

Suppression of miR-93-5p inhibits high-risk HPV-positive cervical cancer progression via targeting of BTG3.

This study explores the role of miR-93-5p in high-risk HPV-positive (HR-HPV) cervical cancer by targeting of BTG3. Cervical tissues were collected from 332 patients with conditions of chronic cervicitis (n = 42), low-grade cervical intraepithelial neoplasia (CIN I, n = 51), CIN II (n = 49), CIN III (n = 43), cervical cancer (n = 90), and normal cervical tissues (n = 57). HR-HPV DNA was detected by Hybrid Capture 2, and the expressions of miR-93-5p and BTG3 were determined by qRT-PCR and Western blot. The target relationship between miR-93-5p and BTG3 was verified by dual-luciferase reporter gene assay. HPV-positive cervical cancer cells (CaSki and HeLa) were divided into control, NC, inhibitor, BTG3, and mimic + BTG3 groups. CCK-8, Annexin V-APC/PI, and Transwell assays were applied to evaluate cell biological activities. MiR-93-5p was positively related but BTG3 was inversely related to HR-HPV infection. Additionally, miR-93-5p expression was negatively correlated with BTG3 expression in cervical cancer tissues infected with HR-HPV. HPV-positive cervical cancer cells showed higher miR-93-5p and lower BTG3 levels than negative cells. CaSki and HeLa cells in the inhibitor group showed increased BTG3 compared with the control group. After transfection with miR-93-5p inhibitor or BTG3 activation plasmid, proliferation and metastasis were inhibited, but apoptosis was promoted. The mimic + BTG3 group showed increased cell proliferation and metastasis but decreased cell apoptosis compared with the BTG3 group. Upregulated miR-93-5p was positively related but downregulated BTG3 was inversely related to HR-HPV infection, and inhibition of miR-93-5p may have blocked HPV-positive cervical cancer development by targeting of BTG3.

Upregulation of MTA1 expression by human papillomavirus infection promotes CDDP resistance in cervical cancer cells via modulation of NF-κB/APOBEC3B cascade.

Compelling evidence establishes the etiological role of viral proteins E6 and E7 of high-risk human papillomaviruses (HPV) in cervical carcinogenesis, but their contribution in chemoresistance that leads to advanced metastatic lesions remains poorly defined. Since metastasis-associated protein 1 (MTA1) upregulation and augmentation of APOBEC3B expression are both strongly associated with cervical cancer (CCa) development, and both molecules have been shown to be functionally associated with NF-κB pathway, we therefore sought to investigate the potential mechanistic link between MTA1, APOBEC3B and NF-κB during the pathogenesis of cisplatin (CDDP) resistance in HPV-positive CCa cells. MTA1 expression was assessed in HPV-transfected CCa cells using quantitative RT-PCR and immunoblotting. Effects of MTA1 deregulation on CDDP chemosensitivity in CCa cells were determined by measuring cell viability, apoptosis and in vivo oncogenic capacity. Finally, we studied the transcriptional regulation of the antiviral DNA cytosine deaminase APOBEC3B by MTA1 using multiple approaches including DNA deaminase activity assay, luciferase reporter assay, chromatin immunoprecipitation, co-immunoprecipitation and transient/stable transfection, at the molecular and functional levels. Expression levels of MTA1 were significantly induced in HPV-positive CCa cells. Transduction experiments showed that the E6 oncoprotein alone was sufficient to cause MTA1 upregulation. Moreover, MTA1 knockdown potentiated CDDP sensitivity in highly metastatic CCa cells. Mechanistically, MTA1 acted as an indirect upstream modulator of APOBEC3B transcription during the pathogenesis of CDDP chemoresistance. HPV-mediated stimulation of APOBEC3B expression was accompanied by the enhanced recruitment of Iκκ α/β and p65 to the NF-κB consensus sites in the APOBEC3B promoter, and this recruitment was substantially abrogated by MTA1 siRNA treatment. These findings reveal an obligatory coregulatory role of MTA1 in the indirect regulation of APOBEC3B expression via classical NF-κB pathway, and also suggest that inhibition of MTA1/NF-κB/APOBEC3B cascade may be repositioned to suppress cancer mutagenesis, dampen tumor evolution, and decrease the probability of adverse outcomes from CDDP resistance in CCa.

Proapoptotic activities of Oroxylum indicum leave extract in HeLa cells

Objective: To examine the proapoptotic properties of Oroxylum indicum methanol extract on cervical cancer cells. Methods: Methylene blue assay was used to determine the IC50 value of the extract. Western blotting assays were done to analyze the expression of HPV oncoproteins (HPV18 E6 and E7) and apoptotic molecules (caspase-3 and caspase-8). Reverse transcriptase PCR assays were performed to determine genetic alteration of tumor suppressors p53 and pRb and apoptosis markers Fas and FasL. Enzyme-linked immunosorbent assay (ELISA) was done to determine the expression of cytokine levels (IL-6 and IL-12). Results: The determination of IC50 value indicated a higher anti-proliferative activity of the extract compared to cisplatin. After 24 hours of treatment, Western blot analysis showed that treated HeLa cells exhibited a significant down-regulation of HPV18 oncoproteins E6 and E7, and a significant induction of caspase-8 and caspase-3 activation level. Meanwhile, the mRNA expressions of p53, pRb, Fas and FasL were significantly upregulated in treated cells. Moreover, ELISA showed an increased IL-12 and decreased IL-6 production after Oroxylum indicum treatment. Conclusions: The methanol extract of Oroxylum indicum has an anti-proliferative activity and proapoptotic potential. It induces localized-immunity improvements by altering cytokine production in HPV-positive cervical cancer cells.

Juglone Inhibits Proliferation of HPV-Positive Cervical Cancer Cells Specifically.

Human papillomaviruses (HPVs), such as HPV 16 and HPV 18 are related to cervical cancer. Therefore, it is important to inhibit HPV-positive cervical cancer for treating cervical cancer. This study is aiming at investigating the proposed molecular mechanism, which underlies the antineoplastic potential of the aqueous extract of juglone of HPV-positive cervical cancer cells. According to the results, it is showed that, juglone prohibited HPV positive cervical cancer cells' growth through dose-dependent way. Nevertheless, when pin 1 was knocked down, the proliferation inhibition reduced. The detection of apoptosis and cell cycle also illustrated that juglone influenced HPV positive cells. Western blot expressed the influence mechanism that it affected the B-cell lymphoma 2 (Bcl-2) family and later activated the Caspase-depended apoptosis way. It is contributable for this study to understand the mechanism of inhibiting HPV positive cells by juglone and it also provides an effective strategy for the application of it in the future.

CRISPR/Cas9-mediated cervical cancer treatment targeting human papillomavirus E6.

High-risk human papillomavirus (HPV) is a common cause of cervical cancer. HPV E6 oncoprotein promotes the degradation of host tumor suppressor gene p53, leading to the development of tumors. Therapeutic strategies that specifically target E6, which is constitutively expressed in tumors and is not present in normal tissues, may be highly effective and safe. CRISPR-CRISPR associated protein 9 (Cas9) is one of the genome editing technologies that has recently garnered attention, and is used to knockout target gene expression. By combining cervical cancer cell lines engineered to constitutively express Cas9 and an adeno-associated virus (AAV) vector carrying a single guide (sg) RNA targeting E6 (AAV-sgE6), the present study sought to investigate the effects of this novel therapeutic approach on cervical cancer. The Cas9 gene was transfected into three high-risk HPV-positive cervical cancer cell lines (HeLa, HCS-2, and SKG-I) to establish cell lines that constitutively expressed Cas9. Using these cell lines, genetic mutations and their frequencies, as well as the levels of protein expression, apoptosis and cell proliferation were examined in vitro. In addition, the effects of AAV-sgE6 were examined in a mouse model of cervical cancer in vivo by a single administration of AAV-sgE6 directly into subcutaneous tumors. The results demonstrated that multiple mutations occurred frequently in the targeted E6 genomic sequence in cervical cancer cells transduced with AAV-sgE6. In addition, these AAV-sgE6-transduced cells had reduced expression of E6, increased expression of p53, increased apoptosis and their growth was suppressed in a concentration-dependent manner. Furthermore, subcutaneous tumor growth was significantly suppressed in vivo following intratumoral administration of AAV-sgE6, and adverse events due to AAV-sgE6 administration were not observed. Collectively, the present results indicated that targeting E6 expression in high-risk HPV by CRISPR-Cas9 is a highly specific and effective strategy that may be effective in treating patients with cervical cancer.

Realgar, a traditional Chinese medicine, induces apoptosis of HPV16-positive cervical cells through a HPV16 E7-related pathway.

PurposeIn this study, we investigated the effect of Realgar on the apoptosis of HPV16-positive cervical cells in vitro.MethodsThe effect of Realgar on the apoptosis of HPV16-positive cervical cells was investigated by annexin V-fluorescein isothiocyanate/propidium iodide staining and growth inhibition assays using HPV16-positive cervical cancer cell line SiHa and HPV16-positive immortalized cervical epithelial cell line S12. The expression of genes was measured by real-time PCR, and the expression of corresponding proteins was detected by Western blotting. The adhesion and invasion of cells were detected by adhesion assay and Transwell invasion assay, respectively.ResultsThe Realgar inhibited the proliferation and induced the apoptosis of SiHa and S12 cells in a dose- and time-dependent manner. The Realgar suppressed the expression of HPV16 E7 and caspase-3. The Realgar suppressed the adhesion and invasion of both cells.ConclusionThe Realgar induced apoptosis, inhibited the proliferation of HPV16-positive cell lines through a HPV16 E7-dependent pathway, and inhibited cell adhesion and invasion.

ALA-PDT promotes HPV-positive cervical cancer cells apoptosis and DCs maturation via miR-34a regulated HMGB1 exosomes secretion.

5-Aminolevulinic acid photodynamic therapy(ALA-PDT) has been widely used for cervical cancer treatment, but the mechanisms are still not fully delineated. Here we showed that ALA-PDT significantly upregulated HMGB1 while downregulated miR-34a expression levels in cervical cancer tissues, and the percentages of mature DCs(mDCs) were increased in ALA-PDT treated patients' peripheral blood. After treating HPV-positive Hela, SiHa, Caski and HPV-negative C33 A cervical cancer cell lines with ALA-PDT, HPV-positive cells' proliferative ability was significantly inhibited and apoptosis rates were elevated, while no significant changes were found in HPV-negative C33 A cell line. Most importantly, in HPV-positive cells, we found that miR-34a were downregulated in cytoplasm, and both cytoplasm and exosome HMGB1 were significantly elevated comparing to cancer cells without ALA-PDT treatment, and it could be reversed by miR-34a mimic transfection, which indicated that HPV infection and miR-34a downregulation might be vital for ALA-PDT treatment. Based on the HMGB1 is the potential target of miR-34a and an inverse correlation between miR-34a and HMGB1 in ALA-PDT treated cancer tissues, we verified that HMGB1 could be targeted and downregulated by miR-34a mimic, and ALA-PDT promotes HMGB1 secretion by inhibiting miR-34a expression. By co-culturing cervical cancer cell lines with immature DCs(imDCs) in the Transwell systems, we found that ALA-PDT induced HMGB1 exosomes could promote DCs maturation, which could be reversed by silencing HMGB1 in HPV-positive cervical cancer cells. In vivo animal experiments also proved that ALA-PDT inhibited tumor growth in tumor bearing mice, which was reversed by co-transfected with miR-34a mimic or silencing HMGB1 in HPV-positive cells. Hence we concluded that ALA-PDT treatment specifically inhibited HPV-positive cervical cancer cells' proliferative ability, promoted cell apoptosis and modulated DCs maturation by regulating miR-34a mediated HMGB1 exosomes secretion.