Elucidation of Rutin role in inducing caspase dependent apoptosis via HPV-E6 and E7 downregulation in cervical cancer HeLa cells

Over the recent few years rutin has gained wider attention in exhibiting inhibitory potential against several oncotargets for inducing apoptotic and antiproliferative activity in several human cancer cells. Several deregulated signaling pathways are implicated in cancer pathogenesis. Therefore we have inclined our research towards exploring the anticancerous efficacy of a very potent phytocompound for modulating the incontinent expression of these two crucial E6 and E7 oncogenes. Further, inhibitory efficacy of rutin against HPV-E6 AND E7 oncoproteins in cervical cancer has not been elucidated yet. This research addresses the growth inhibitory efficacy of rutin against E6 and E7 oncoprotein in HeLa cells, which is known to inactivate several tumor suppressor protein such as p53 and pRB. Rutin treatment exhibited reduced cell viability with increased cell accumulation in G0/G1 phase of cell cycle in Hela cell lines. Additionally rutin treatment has also led to downregulation of E6 and E7 expression associated with an increased expression of p53 and pRB levels. This has further resulted in enhanced Bax expression and decreased Bcl-2 expression releasing cytochrome c into cytosol followed by caspase cascade activation with cleavage of caspase-3 caspase-8 and caspase-9. Further, in silico studies have also supported our in vitro findings by exhibiting significant binding energy against selected target oncoproteins. Therefore, our research findings might recommend rutin as one of the potent drug candidate in cervical cancer management via targeting two crucial oncoproteins associated with viral progression.

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In Silico Approaches: A Way to Unveil Novel Therapeutic Drugs for Cervical Cancer Management

Pharmaceuticals ◽

10.3390/ph14080741 ◽

2021 ◽

Vol 14 (8) ◽

pp. 741

Author(s):

Diana Gomes ◽

Samuel Silvestre ◽

Ana Paula Duarte ◽

Aldo Venuti ◽

Christiane P. Soares ◽

...

Keyword(s):

Cervical Cancer ◽

Small Molecules ◽

In Silico ◽

Access To Health Services ◽

Cancer Management ◽

Hpv E6 ◽

Therapeutic Drugs ◽

E6 Oncoprotein ◽

Access To Health ◽

E6 And E7

Cervical cancer (CC) is the fourth most common pathology in women worldwide and presents a high impact in developing countries due to limited financial resources as well as difficulties in monitoring and access to health services. Human papillomavirus (HPV) is the leading cause of CC, and despite the approval of prophylactic vaccines, there is no effective treatment for patients with pre-existing infections or HPV-induced carcinomas. High-risk (HR) HPV E6 and E7 oncoproteins are considered biomarkers in CC progression. Since the E6 structure was resolved, it has been one of the most studied targets to develop novel and specific therapeutics to treat/manage CC. Therefore, several small molecules (plant-derived or synthetic compounds) have been reported as blockers/inhibitors of E6 oncoprotein action, and computational-aided methods have been of high relevance in their discovery and development. In silico approaches have become a powerful tool for reducing the time and cost of the drug development process. Thus, this review will depict small molecules that are already being explored as HR HPV E6 protein blockers and in silico approaches to the design of novel therapeutics for managing CC. Besides, future perspectives in CC therapy will be briefly discussed.

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Cytotoxicity Effect of Quercus infectoria Based Vaginal Cream on Hela Cells and its Preliminary in Vivo Toxicity Evaluation Towards Female Rats

IIUM Medical Journal Malaysia ◽

10.31436/imjm.v17i2.276 ◽

2020 ◽

Vol 17 (2) ◽

Author(s):

Hazwani MY ◽

Hasmah A ◽

Wan Amir Nizam WA

Keyword(s):

Cervical Cancer ◽

Rat Model ◽

Hela Cells ◽

Reproductive Tract ◽

Radical Scavenging ◽

Female Rat ◽

Hpv E6 ◽

Vaginal Cream ◽

E6 And E7 ◽

Dpph Radical Scavenging

Introduction: Cervical cancer is the third leading cause of cancer death among females in less developed countries. Drugs used in the treatment of cervical cancer were reported to exert a cytotoxic effect on the normal cells. This study aimed to determine the effectiveness of Quercus infectoria (QI) vaginal cream towards cervical cancer cell, HeLa and its toxicity effect on the female rat model. Methods: MTT assays were utilized to determine the median concentration (IC50) for cell cytotoxicity of QIA and QI vaginal cream against cervical cancer cells, HeLa. Expression of HPV E6 and E7 protein in HeLa cells treated with QI vaginal cream for 24 hours were conducted by Western blot analysis. In separate experiments, the toxicity of QI vaginal cream on a lower reproductive tract of the female rat model has been assessed by histopathological examination after application for three weeks. The antioxidant activity of QIA extract and QI vaginal cream were assessed by DPPH radical scavenging assay. Results: A moderate cytotoxicity activity exerted by QIA extract and QI vaginal cream against HeLa cell with IC50 values of 13.90 ± 2.27, and 20.80 ± 1.94 respectively. Furthermore, QI vaginal cream suppressed the expression of HPV E6 and E7. Daily application of QI vaginal did not exert any inflammation to the vaginal mucosa and cervix. QIA extract and QI vaginal cream demonstrated high DPPH radical scavenging activity. Conclusion: Formulated QI vaginal cream has cytotoxic effect on HeLa cells without causing an adverse effect on the lower reproductive tract in female rat model.

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Complementary Targeting of Rb Phosphorylation and Growth in Cervical Cancer Cell Cultures and a Xenograft Mouse Model by SHetA2 and Palbociclib

Cancers ◽

10.3390/cancers12051269 ◽

2020 ◽

Vol 12 (5) ◽

pp. 1269 ◽

Cited By ~ 1

Author(s):

Amy L. Kennedy ◽

Rajani Rai ◽

Zitha Redempta Isingizwe ◽

Yan Daniel Zhao ◽

Stanley A. Lightfoot ◽

...

Keyword(s):

Cervical Cancer ◽

Cyclin D1 ◽

Cell Lines ◽

Cancer Cell ◽

Xenograft Model ◽

Cervical Cancer Cell ◽

Chi Square ◽

Western Blots ◽

Hpv E6 ◽

E6 And E7

Cervical cancer is caused by high-risk human papillomavirus (HPV) types and treated with conventional chemotherapy with surgery and/or radiation. HPV E6 and E7 proteins increase phosphorylation of retinoblastoma (Rb) by cyclin D1/cyclin dependent kinase (CDK)4/6 complexes. We hypothesized that cyclin D1 degradation by the SHetA2 drug in combination with palbociclib inhibition of CDK4/6 activity synergistically reduces phosphorylated Rb (phospho-Rb) and inhibits cervical cancer growth. The effects of these drugs, alone, and in combination, were evaluated in SiHa and CaSki HPV-positive and C33A HPV-negative cervical cancer cell lines using cell culture, western blots and ELISA, and in a SiHa xenograft model. Endpoints were compared by isobolograms, ANOVA, and Chi-Square. In all cell lines, combination indexes documented synergistic interaction of SHetA2 and palbociclib in association SHetA2 reduction of cyclin D1 and phospho-Rb, palbociclib reduction of phospho-Rb, and enhanced phospho-Rb reduction upon drug combination. Both drugs significantly reduced phospho-Rb and growth of SiHa xenograft tumors as single agents and acted additively when combined, with no evidence of toxicity. Dilated CD31-negative blood vessels adjacent to, or within, areas of necrosis and apoptosis were observed in all drug-treated tumors. These results justify development of the SHetA2 and palbociclib combination for targeting phospho-Rb in cervical cancer treatment.

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Anticancer Effect of Natural Product Sulforaphane by Targeting MAPK Signal through miRNA-1247-3p in Human Cervical Cancer Cells

Biointerface Research in Applied Chemistry ◽

10.33263/briac111.79437972 ◽

2020 ◽

Vol 11 (1) ◽

pp. 7943-7972

Keyword(s):

Cervical Cancer ◽

Flow Cytometry ◽

Western Blot ◽

Hela Cells ◽

Mapk Pathway ◽

Mapk Signaling ◽

Cell Counting ◽

Hela Cell Lines ◽

Cervical Cancer Cells ◽

Induced Apoptosis

The prognosis of cervical cancer remains poor. Sulforaphane, an active ingredient from cruciferous plants, has been identified as a potential anticancer agent in various cancers. However, there is little information about its effect on cervical cancer. Here, we conducted a present study to uncover the effect and the potential mechanisms of sulforaphane on cervical cancer. HeLa cells were treated with sulforaphane, and cell proliferation and apoptosis were assessed by Cell Counting Kit-8, Western blot, flow cytometry, and immunofluorescence. Then, next-generation sequencing (NGS) and bioinformatics tools were used to analyze mRNA-seq, miRNA-seq, and potential pathways. Finally, qRT-PCR, Cell Counting Kit-8, flow cytometry, small RNAs analysis, and Western blot were performed to evaluate the biological function of miR1247-3p and MAPK pathway in HeLa cell lines. Sulforaphane significantly suppressed the viability and induced apoptosis of HeLa cells. NGS and bioinformatics analysis showed sulforaphane exerted its anti-tumor activities through miR1247-3p and the MAPK signaling pathway. Further analysis suggested that sulforaphane could activate MAPK pathway via down-regulating the expression of miR-1247-3p. Sulforaphane inhibited proliferation and promoted apoptosis of HeLa cells via down-regulation of miR-1247-3p and activating the MAPK pathway. These findings provide preliminary experimental evidence for the treatment of cervical cancer with sulforaphane.

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Transcriptome Analysis of Cervical Cancer Exosomes and Detection of HPVE6*I Transcripts in Exosomal RNA

10.21203/rs.3.rs-880218/v1 ◽

2021 ◽

Author(s):

Anjali Bhat ◽

Joni Yadav ◽

Kulbhushan Thakur ◽

Nikita Aggarwal ◽

Arun Chhokar ◽

...

Keyword(s):

Cervical Cancer ◽

Hela Cells ◽

Cell Communication ◽

Cellular Migration ◽

Migration And Invasion ◽

P Value ◽

Rt Pcr ◽

Illumina Hiseq ◽

Kegg Pathways ◽

Hpv E6

Abstract Background Exosomes play a key role in cell-to-cell communication and are integral component of the tumor microenvironment. Recent observations suggest transfer of RNA through tumor-derived exosomes that can potentially translate into regulatory proteins in the recipient cells. Role of cervical cancer-derived exosomes and their transcript cargo is poorly understood. Materials and Methods The total RNA of exosomes from HPV-positive (SiHa and HeLa) and HPV-negative (C33a) cervical cancer cell lines were extracted and the transcripts were estimated using Illumina HiSeq X. Further, validation of HPV transcripts were performed using RT-PCR. Results 3099 transcripts were found to be differentially-exported in HPV-positive vs. HPV-negative exosomes (p value < 0.05). Analysis of top 10 GO terms and KEGG pathways showed enrichment of transcripts belonging to axon guidance and tumor innervation in HPV-positive exosomes. Among top 20 overexpressed transcripts, EVC2, LUZP1 and ANKS1B were the most notable due to their involvement in Hh signaling, cellular migration and invasion, respectively. Further, low levels of HPV-specific reads were detected. RT-PCR validation revealed presence of E6*I splice variant of HPV18 in exosomal RNA of HeLa cells. The E6*I transcripts were consistently retained in exosomes obtained from HeLa cells undergoing 5-FU and cisplatin-induced oxidative stress. Conclusion Our data suggests the enrichment of poly-A RNA transcripts in the exosomal cargo of cervical cancer cells, which includes pro-tumorigenic cellular RNA and viral transcripts such as HPV E6, which may have clinical utility as potential exosomal biomarkers of cervical cancer.

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The potency of chitosan-based Pinus merkusii bark extract nanoparticles as anti-cancer on HeLa cell lines

Veterinary World ◽

10.14202/vetworld.2019.1616-1623 ◽

2019 ◽

Vol 12 (10) ◽

pp. 1616-1623

Author(s):

Annise Proboningrat ◽

Amaq Fadholly ◽

Regina Purnama Dewi Iskandar ◽

Agung Budianto Achmad ◽

Fedik Abdul Rantam ◽

...

Keyword(s):

Cell Cycle ◽

Cervical Cancer ◽

Hela Cell ◽

Hela Cells ◽

Therapeutic Potential ◽

Bark Extract ◽

Cytotoxicity Test ◽

Caspase 9 ◽

Hela Cell Lines ◽

Pinus Merkusii

Background and Aim: Cervical cancer accounts for the fourth as a cause of death from cancer in women worldwide, with more than 85% of events and deaths occurring in developing countries. The main problems of chemotherapy are the lack of selectivity and drug resistance. This study aimed to investigate the signal transduction of chitosan-based Pinus merkusii bark extract nanoparticles (Nano-PMBE) as an anticancer on HeLa cell line. Materials and Methods: Nano-PMBE was prepared based on the ionic gelation method. Its anticancer activities in HeLa cells were investigated through cytotoxicity test, cell cycle, and apoptosis analysis. The expression of p53 and caspase-9 was also observed. Results: The results showed that Nano-PMBE has a size of 394.3 nm. Meanwhile, the Nano-PMBE was cytotoxic to HeLa cells ( IC50 of 384.10 μg/ml), caused G0/G1 phase arrest and cell apoptosis in HeLa cells. Besides, the expression of p53 and caspase-9 has increased. Conclusion: The results showed a notable anticancer effect of Nano-PMBE by arresting the cell cycle and inducing apoptosis in HeLa cells, suggesting that it might have therapeutic potential for cervical cancer. Further research is needed to find out more about the anticancer mechanism of Nano-PMBE in HeLa cells to in vivo and clinical studies.

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E6-mediated activation of JNK drives EGFR signalling to promote proliferation and viral oncoprotein expression in cervical cancer

Cell Death and Differentiation ◽

10.1038/s41418-020-00693-9 ◽

2020 ◽

Author(s):

Ethan L. Morgan ◽

James A. Scarth ◽

Molly R. Patterson ◽

Christopher W. Wasson ◽

Georgia C. Hemingway ◽

...

Keyword(s):

Cervical Cancer ◽

Cell Proliferation ◽

Cancer Cells ◽

Human Papillomaviruses ◽

Signalling Pathway ◽

Viral Oncoprotein ◽

Hpv E6 ◽

Cervical Cancer Cells ◽

E6 And E7 ◽

Novel Therapeutic

AbstractHuman papillomaviruses (HPV) are a major cause of malignancy worldwide, contributing to ~5% of all human cancers including almost all cases of cervical cancer and a growing number of ano-genital and oral cancers. HPV-induced malignancy is primarily driven by the viral oncogenes, E6 and E7, which manipulate host cellular pathways to increase cell proliferation and enhance cell survival, ultimately predisposing infected cells to malignant transformation. Consequently, a more detailed understanding of viral-host interactions in HPV-associated disease offers the potential to identify novel therapeutic targets. Here, we identify that the c-Jun N-terminal kinase (JNK) signalling pathway is activated in cervical disease and in cervical cancer. The HPV E6 oncogene induces JNK1/2 phosphorylation in a manner that requires the E6 PDZ binding motif. We show that blockade of JNK1/2 signalling using small molecule inhibitors, or knockdown of the canonical JNK substrate c-Jun, reduces cell proliferation and induces apoptosis in cervical cancer cells. We further demonstrate that this phenotype is at least partially driven by JNK-dependent activation of EGFR signalling via increased expression of EGFR and the EGFR ligands EGF and HB-EGF. JNK/c-Jun signalling promoted the invasive potential of cervical cancer cells and was required for the expression of the epithelial to mesenchymal transition (EMT)-associated transcription factor Slug and the mesenchymal marker Vimentin. Furthermore, JNK/c-Jun signalling is required for the constitutive expression of HPV E6 and E7, which are essential for cervical cancer cell growth and survival. Together, these data demonstrate a positive feedback loop between the EGFR signalling pathway and HPV E6/E7 expression, identifying a regulatory mechanism in which HPV drives EGFR signalling to promote proliferation, survival and EMT. Thus, our study has identified a novel therapeutic target that may be beneficial for the treatment of cervical cancer.

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Flax seed oil reduced tumor growth, modulated immune responses and decreased HPV E6 and E7 oncoprotein expression in a murine model of ectopic cervical cancer

Prostaglandins & Other Lipid Mediators ◽

10.1016/j.prostaglandins.2019.04.002 ◽

2019 ◽

Vol 143 ◽

pp. 106332 ◽

Cited By ~ 2

Author(s):

Rashmi Deshpande ◽

Prerna Raina ◽

Kavita Shinde ◽

Prakash Mansara ◽

Manjiri Karandikar ◽

...

Keyword(s):

Cervical Cancer ◽

Immune Responses ◽

Tumor Growth ◽

Murine Model ◽

Seed Oil ◽

Flax Seed ◽

E7 Oncoprotein ◽

Hpv E6 ◽

E6 And E7

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HPV-targeted tumor-infiltrating lymphocytes for cervical cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2014.32.18_suppl.lba3008 ◽

2014 ◽

Vol 32 (18_suppl) ◽

pp. LBA3008-LBA3008 ◽

Cited By ~ 2

Author(s):

Christian S. Hinrichs ◽

Sanja Stevanovic ◽

Lindsey Draper ◽

Robert Somerville ◽

John Wunderlich ◽

...

Keyword(s):

Clinical Trial ◽

Cervical Cancer ◽

T Cells ◽

Tumor Infiltrating Lymphocytes ◽

The Other ◽

High Dose ◽

Complete Regression ◽

Hpv E6 ◽

E6 And E7 ◽

Infiltrating Lymphocytes

LBA3008 Background: Adoptive T-cell therapy (ACT) is a promising cancer treatment modality with potentially broad application. It is not known if ACT can mediate regression of carcinomas, the most common solid tumors in humans. We studied carcinoma of the uterine cervix, a virally induced malignancy that constitutively expresses the HPV E6 and E7 oncoproteins, as a model cancer to test if ACT can mediate regression of an epithelial malignancy. Methods: We initiated a clinical trial to treat metastatic HPV+ cancers with tumor-infiltrating lymphocytes (TIL) selected for HPV E6- and E7-reactivity (HPV-TIL). Patients from the cervical cancer cohort are reported here. HPV-TIL infusion was preceded b y non-myeloablative conditioning and followed by high-dose bolus aldesleukin. HPV-reactivity was assessed by ELISPOT, IFN-gamma production, and CD137 expression assays. Results: Nine patients were treated on the study. They received a median of 81 x 109 T cells (range 33 to 159 x 109) as a single infusion. The infused cells possessed reactivity against high-risk HPV E6 and/or E7 in 6/8 patients. The two patients with no HPV reactivity did not respond to treatment. 3/6 patients with HPV reactivity demonstrated objective tumor responses by RECIST (1 PR and 2 CR). One patient had a 39% best response. Two patients with widespread metastases had complete tumor responses that are ongoing 18 and 11 months after treatment. One patient with a complete response had a chemotherapy-refractory HPV-16+ squamous cell carcinoma and the other a chemoradiation-refractory HPV-18+ adenocarcinoma. Both patients demonstrated prolonged repopulation with HPV-reactive T cells following treatment. Increased frequencies of HPV-specific T cells were detectable after 13 months in one patient and 6 months in the other. Two patients with HPV-reactive TIL that did not respond to treatment did not display repopulation with HPV-reactive T cells. Conclusions: HPV-TIL can mediate durable, complete regression of metastatic cervical cancer. Continued investigation of HPV-TIL for cervical cancer, and possibly other HPV+ malignancies, is warranted. Cellular therapy can mediate complete regression of an epithelial malignancy. Clinical trial information: NCT01585428.

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