Whole Genome Assembly of Human Papillomavirus by Nanopore Long-Read Sequencing

Human papillomavirus (HPV) is a causal agent for most cervical cancers. The physical status of the HPV genome in these cancers could be episomal, integrated, or both. HPV integration could serve as a biomarker for clinical diagnosis, treatment, and prognosis. Although whole-genome sequencing by next-generation sequencing (NGS) technologies, such as the Illumina sequencing platform, have been used for detecting integrated HPV genome in cervical cancer, it faces challenges of analyzing long repeats and translocated sequences. In contrast, Oxford nanopore sequencing technology can generate ultra-long reads, which could be a very useful tool for determining HPV genome sequence and its physical status in cervical cancer. As a proof of concept, in this study, we completed whole genome sequencing from a cervical cancer tissue and a CaSki cell line with Oxford Nanopore Technologies. From the cervical cancer tissue, a 7,894 bp-long HPV35 genomic sequence was assembled from 678 reads at 97-fold coverage of HPV genome, sharing 99.96% identity with the HPV sequence obtained by Sanger sequencing. A 7904 bp-long HPV16 genomic sequence was assembled from data generated from the CaSki cell line at 3857-fold coverage, sharing 99.99% identity with the reference genome (NCBI: U89348). Intriguingly, long reads generated by nanopore sequencing directly revealed chimeric cellular–viral sequences and concatemeric genomic sequences, leading to the discovery of 448 unique integration breakpoints in the CaSki cell line and 60 breakpoints in the cervical cancer sample. Taken together, nanopore sequencing is a unique tool to identify HPV sequences and would shed light on the physical status of HPV genome in its associated cancers.

Cholecalciferol Inhibits Cell Growth and Induces Apoptosis in the CaSki Cell Line

Vitamin D has displayed anti-cancer actions in numerous in vitro studies. Here, we investigated the anti-cancer actions of cholecalciferol, a vitamin D precursor, on a metastatic cervical cancer cell line, namely, CaSki. Experimental cultures were incubated for 72 h and treated with cholecalciferol (10–1000 ng/mL). In the present study, cell count, viability, proliferation and cell cycle were analyzed by a crystal violet assay, trypan blue assay, Ki67 proliferation, and a cell cycle assay, respectively. Biomarkers of apoptosis, necrosis, and autophagic cell death were measured by the Caspase 3/7 and Annexin V/7-AAD Muse™ assays, a LC3-II assay, and a lactate dehydrogenase release assay, respectively. The ultrastructural features of cell death were assessed by transmission electron microscopy. A statistical analysis was performed using a one-way ANOVA and Bonferroni’s post-hoc analysis test, and p < 0.05 is considered statistically significant here. The results identify statistical decreases in cell count and viability at high-dose treatments (100 and 1000 ng/mL). In addition, significant increases in apoptotic biochemical markers and apoptotic ultrastructure are shown to be present at high-dose treatments. In conclusion, high-dose cholecalciferol treatments inhibit cell count and viability, which are both mediated by apoptotic induction in the CaSki cell line.

THE APOPTOTIC EFFECT OF RESVERATROL ON HUMAN PAPILLOMA VIRUS POSITIVE CASKI CELL-LINE: A TIME AND DOSE DEPENDENCE STUDY.

Background & objectives: Resveratrol (trans-3,4,5’ –trihydroxystilbene) is an active compound in food, such as redgrapes, peanuts, and berries. A body of evidence shows that resveratrol is able to inhibit thegrowth of many cancers such as leukemia, breast cancer and primary brain tumors. The objective of the present study was to demonstrate the dose and time dependent apoptotic effect of Resveratrol on Human Caski cell line. Methods: We used cell viability assays like MTT and Trypan blue assays to demonstrate the inhibition of cancer cell proliferation in a dose and time dependent manner. Results: Our results have shown that Resveratrol inhibited proliferation of Caski cells in a concentration and time dependent manner in 48 hr cultures. No significant effect was observed in 24 hour cultures of cell viability assays. Interpretation & conclusions: The results indicated that Resveratrol and NAC inhibited cell growth in Caski cells as a function of dose and time. Keywords: Resveratrol , Cervical cancer, apoptosis

Phototoxic effects of pyropheophorbide-a from chlorophyll-a on cervical cancer cells

Photodynamic therapy (PDT) is a promising modality in both the curative and palliative treatment against a variety of experimental and naturally occurring human cancers. At present, chlorophyll a derivatives are extensively used for the synthesis of photosensitizers (PSs) for PDT of tumors. In the present study, chlorophyll-a was extracted from the blue-green algae Spirulina platensis by refluxing with acetone. The extract was further acid treated to obtain methylpheophorbide-a (MPa), which was then refluxed in collidine and methylpyropheophorbide-a (Mppa) was obtained. After that, Mppa was converted to pyropheophorbide-a (Ppa) by treatment with 50% sulfuric acid. Finally, phototoxicity and dark toxicity of purified Ppa in two different cell lines, TC-1 and CaSki, were examined by MTT assay. The results suggest that Ppa is more toxic to TC-1 cell line than CaSki cell line. In vivo, the photosensitizing efficiency of Ppa was also higher than those of unloaded PS. These results indicate the potential of Ppa in PDT.