9-oxo-ODAs suppresses the proliferation of human cervical cancer cells through the inhibition of CDKs and HPV oncoproteins.

Mucosal human papillomavirus (HPV) subtypes 16 and 18 are causative agents of cervical cancer, a leading cause of cancer-related deaths among women worldwide. In Japan, eggplant calyx is a folk remedy used to treat common warts. 9-oxo-(10E,12E)-octadecadienoic acid, isolated from eggplant calyx, may have antitumor effects. This study investigated the antitumor effects of 9-oxo-(10E, 12Z)-octadecadienoic acid and 9-oxo-(10E,12E)-octadecadienoic acid (9-oxo-ODAs) on human cervical cancer cells. 9-oxo-ODAs suppressed the proliferation of human cervical cancer cell lines (HeLa, and SiHa) in a concentration-dependent manner (IC50 = 25-50 µM). FCM analysis revealed that 9-oxo-ODAs induced apoptosis. Transcriptome, proteomics, and enrichment analyses revealed that treatment with 9-oxo-ODAs significantly altered the cell cycle and p53 pathways and decreased cyclin-dependent kinase 1 (CDK1) protein expression. Real-time PCR analysis demonstrated that 9-oxo-ODAs reduced CDK1 mRNA expression in a concentration-dependent manner. In vitro, 9-oxo-ODAs reduced the HPV oncoprotein expression. In ex vivo human cervical cancer tissues, 9-oxo-ODAs decreased CDK1 expression and increased cleaved caspase 3, an apoptosis marker. Further, 9-oxo-ODAs showed the potential to suppressed metastatic formation and growth of cervical cancer in vivo. These findings suggest that 9-oxo-ODAs induce cell cycle arrest and apoptosis in HPV-positive human cervical cancer cells, and this process involves CDK1. Consequently, 9-oxo-ODAs may be potential therapeutic agents for cervical cancer.

Green Tea Polyphenols Cause Apoptosis and Autophagy in HPV-16 Subgene-Immortalized Human Cervical Epithelial Cells via the Activation of the Nrf2 Pathway.

Infection with human papillomavirus (HPV) is relatively common and certain high-risk HPV strains can induce epithelial dysplasia, increasing the risk of cervical cancer. Green tea polyphenol (GTP) preparations exhibit diverse anti-inflammatory, antioxidative, and antitumor properties In Vitro and In Vivo. Topical GTP application has been recommended as a treatment for genital warts, but the effect of GTP treatment on HPV infection and HPV-associated cancer remains to be established. The present study aimed to explore the mechanism by which GTP affected HPV type 16 (HPV-16)-positive immortalized human cervical epithelial cells. Survival, apoptosis, and autophagocytosis of these cells following GTP treatment was assessed using CCK-8 assay, flow cytometry, and monodansylcadaverine (MDC) staining. These cells were further transfected with an shRNA specific for Nrf2 to generate stable Nrf2-knockdown cells. The levels of Caspase-3, Bcl-2, Bax, P53, Rb, HPV-16 E6, HPV-16 E7, P62, Beclin1 and LC3B were determined via Western blotting. These analyses revealed that GTP treatment induced autophagy and apoptosis in HPV-16-positive cells, while Nrf2 gene knockdown reversed GTP-induced autophagic and apoptotic effects. Together, these results suggested that GTP could alleviate HPV infection and HPV-associated precancerous lesions In Vitro by regulating the Nrf2 pathway, highlighting the therapeutic potential of GTP in treating HPV infection.

Inducible expression of human papillomavirus-16 L1 capsomeres in the plastomes of Nicotiana tabacum: Transplastomic plants develop normal flowers and pollen.

Human papillomavirus type-16 (HPV-16) is the major HPV type involved in causing cervical cancer among women. The disease burden is high in developing and underdeveloped countries. Previously, the constitutive expression of HPV-16 L1 protein led to male sterility in transplastomic tobacco plants. Here, the HPV-16 L1 gene was expressed in chloroplasts of Nicotiana tabacum under the control of an ethanol-inducible promoter, trans-activated by nucleus-derived signal peptide. Plants containing nuclear component were transformed with transformation vector pEXP-T7-L1 by biolistic gun. The transformation and homoplasmic status of transformed plants was verified by polymerase chain reaction and Southern blotting, respectively. Protein was induced by spraying 5% ethanol for 7 consecutive days. The correct folding of L1 protein was confirmed by antigen-capture ELISA using a conformation-specific antibody. The L1 protein accumulated up to 3 µg/g of fresh plant material. The L1 protein was further purified using affinity chromatography. All transplastomic plants developed normal flowers and produced viable seeds upon self-pollination. Pollens also showed completely normal structure under light microscope and scanning electron microscopy. These data confirm the use of the inducible expression as plant-safe approach for expressing transgenes in plants, especially those genes that cause detrimental effects on plant growth and morphology.

Selenium atom on phosphate enhances specificity and sensitivity of DNA polymerization and detection.

DNA polymerization is of high specificity in vivo. However, its specificity is much lower in vitro, which limits advanced applications of DNA polymerization in ultrasensitive nucleic acid detection. Herein, we report a unique mechanism of single selenium-atom modified dNTP (dNTPαSe) to enhance polymerization specificity. We have found that both dNTPαSe (approximately 660 fold) and Se-DNA (approximately 2.8 fold) have lower binding affinity to DNA polymerase than canonical ones, and the Se-DNA duplex has much lower melting-temperature (Tm) than the corresponding canonical DNA duplex. The reduced affinity and Tm can destabilize the substrate-primer-template-enzyme assembly, thereby largely slowing down the mismatch of DNA polymerization and enhancing the amplification specificity and in turn detection sensitivity. Furthermore, the Se-strategy enables us to develop the selenium enhanced specific isothermal amplification (SEA) for nucleic acid detection with high specificity and sensitivity (up to detection of single-digit copies), allowing convenient detection of clinical HPV and COVID-19 viruses in the low-copy number. Clearly, we have discovered the exciting mechanism for enhancing DNA polymerization accuracy, amplification specificity and detection sensitivity by SEA, up to two orders of magnitude higher.

Oncogenic HPV promotes the expression of the long noncoding RNA lnc-FANCI-2 through E7 and YY1.

Long noncoding RNAs (lncRNAs) play diverse roles in biological processes, but their expression profiles and functions in cervical carcinogenesis remain unknown. By RNA-sequencing (RNA-seq) analyses of 18 clinical specimens and selective validation by RT-qPCR analyses of 72 clinical samples, we provide evidence that, relative to normal cervical tissues, 194 lncRNAs are differentially regulated in high-risk (HR)-HPV infection along with cervical lesion progression. One such lncRNA, lnc-FANCI-2, is extensively characterized because it is expressed from a genomic locus adjacent to the FANCI gene encoding an important DNA repair factor. Both genes are up-regulated in HPV lesions and in in vitro model systems of HR-HPV18 infection. We observe a moderate reciprocal regulation of lnc-FANCI-2 and FANCI in cervical cancer CaSki cells. In these cells, lnc-FANCI-2 is transcribed from two alternative promoters, alternatively spliced, and polyadenylated at one of two alternative poly(A) sites. About 10 copies of lnc-FANCI-2 per cell are detected preferentially in the cytoplasm. Mechanistically, HR-HPVs, but not low-risk (LR)-HPV oncogenes induce lnc-FANCI-2 in primary and immortalized human keratinocytes. The induction is mediated primarily by E7, and to a lesser extent by E6, mostly independent of p53/E6AP and pRb/E2F. We show that YY1 interacts with an E7 CR3 core motif and transactivates the promoter of lnc-FANCI-2 by binding to two critical YY1-binding motifs. Moreover, HPV18 increases YY1 expression by reducing miR-29a, which targets the 3' untranslated region of YY1 mRNA. These data have provided insights into the mechanisms of how HR-HPV infections contribute to cervical carcinogenesis.

A Potential Herbal Adjuvant Combined With a Peptide-Based Vaccine Acts Against HPV-Related Tumors Through Enhancing Effector and Memory T-Cell Immune Responses.

Viral infection is associated with many types of tumorigenesis, including human papillomavirus (HPV)-induced cervical cancer. The induction of a specific T-cell response against virus-infected cells is desired to develop an efficient therapeutic approach for virus-associated cancer. Chinese herbal medicine (CHM) has a long history in the treatment of cancer patients in Asian countries. Hedyotis diffusa Willd (Bai Hua She She Cao, BHSSC) is frequently used clinically and has been shown to inhibit tumor growth in vitro. However, in vivo data demonstrating the antitumor efficacy of BHSSC are still lacking. We showed that BHSSC induces murine and human antigen-presenting cell (APC) activation via the MAPK signaling pathway and enhances antigen presentation in bone marrow-derived dendritic cells (BMDCs) in vitro. Furthermore, we identified that treatment with BHSSC leads to improved specific effector and memory T-cell responses in vivo. Variant peptide-based vaccines combined with BHSSC improved antitumor activity in preventive, therapeutic, and recurrent HPV-related tumor models. Furthermore, we showed that rutin, one of the ingredients in BHSSC, induces a strong specific immune response against HPV-related tumors in vivo. In summary, we demonstrated that BHSSC extract and its active compound, rutin, can be used as adjuvants in peptide-based vaccines to increase immunogenicity and to bypass the requirement of a conditional adjuvant.

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

PURPOSE: In this study, we investigated the effect of Realgar on the apoptosis of HPV16-positive cervical cells in vitro. METHODS: The 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. RESULTS: The 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. CONCLUSION: The Realgar induced apoptosis, inhibited the proliferation of HPV16-positive cell lines through a HPV16 E7-dependent pathway, and inhibited cell adhesion and invasion.

Hyperthermia Selectively Targets Human Papillomavirus in Cervical Tumors via p53-Dependent Apoptosis.

Human papillomavirus (HPV) is associated with cervical cancer, the third most common cancer in women. The high-risk HPV types 16 and 18 are found in over 70% of cervical cancers and produce the oncoprotein, early protein 6 (E6), which binds to p53 and mediates its ubiquitination and degradation. Targeting E6 has been shown to be a promising treatment option to eliminate HPV-positive tumor cells. In addition, combined hyperthermia with radiation is a very effective treatment strategy for cervical cancer. In this study, we examined the effect of hyperthermia on HPV-positive cells using cervical cancer cell lines infected with HPV 16 and 18, in vivo tumor models, and ex vivo-treated patient biopsies. Strikingly, we demonstrate that a clinically relevant hyperthermia temperature of 42 °C for 1 hour resulted in E6 degradation, thereby preventing the formation of the E6-p53 complex and enabling p53-dependent apoptosis and G2-phase arrest. Moreover, hyperthermia combined with p53 depletion restored both the cell-cycle distribution and apoptosis to control levels. Collectively, our findings provide new insights into the treatment of HPV-positive cervical cancer and suggest that hyperthermia therapy could improve patient outcomes.

Tannins from Hamamelis virginiana bark extract: characterization and improvement of the antiviral efficacy against influenza A virus and human papillomavirus.

Antiviral activity has been demonstrated for different tannin-rich plant extracts. Since tannins of different classes and molecular weights are often found together in plant extracts and may differ in their antiviral activity, we have compared the effect against influenza A virus (IAV) of Hamamelis virginiana L. bark extract, fractions enriched in tannins of different molecular weights and individual tannins of defined structures, including pseudotannins. We demonstrate antiviral activity of the bark extract against different IAV strains, including the recently emerged H7N9, and show for the first time that a tannin-rich extract inhibits human papillomavirus (HPV) type 16 infection. As the best performing antiviral candidate, we identified a highly potent fraction against both IAV and HPV, enriched in high molecular weight condensed tannins by ultrafiltration, a simple, reproducible and easily upscalable method. This ultrafiltration concentrate and the bark extract inhibited early and, to a minor extent, later steps in the IAV life cycle and tannin-dependently inhibited HPV attachment. We observed interesting mechanistic differences between tannin structures: High molecular weight tannin containing extracts and tannic acid (1702 g/mol) inhibited both IAV receptor binding and neuraminidase activity. In contrast, low molecular weight compounds (<500 g/mol) such as gallic acid, epigallocatechin gallate or hamamelitannin inhibited neuraminidase but not hemagglutination. Average molecular weight of the compounds seemed to positively correlate with receptor binding (but not neuraminidase) inhibition. In general, neuraminidase inhibition seemed to contribute little to the antiviral activity. Importantly, antiviral use of the ultrafiltration fraction enriched in high molecular weight condensed tannins and, to a lesser extent, the unfractionated bark extract was preferable over individual isolated compounds. These results are of interest for developing and improving plant-based antivirals.

Wogonin induces apoptosis by suppressing E6 and E7 expressions and activating intrinsic signaling pathways in HPV-16 cervical cancer cells.

Wogonin is a flavonoid compound extracted from Scutellaria baicalensis and is well known as a benzodiazepine receptor ligand with anxiolytic effects. Many recent studies have demonstrated that wogonin modulates angiogenesis, proliferation, invasion, and tumor progress in various cancer tissues. We further explored the mechanism of action of wogonin on cervical cancer cells that contain or lack human papillomavirus (HPV) DNA. Wogonin was cytotoxic to HPV 16 (+) cervical cancer cells, SiHa and CaSki, but not to HPV-negative cells. We demonstrated that wogonin induced apoptosis by suppressing the expressions of the E6 and E7 viral oncogenes in HPV-infected cervical cancer CaSki and SiHa cells. The modulation of p53 and protein retinoblastoma (pRb) were also triggered by the suppression of E6 and E7 expressions. However, p53 was not altered in HPV-negative cervical cancer C33A cells. Moreover, wogonin modulated the mitochondrial membrane potential and the expression of pro- and anti-apoptotic factors such as Bax and Bcl-2. Wogonin also provoked the cleavage of caspase-3, caspase-9, and poly ADP ribose polymerase. After transfection of siRNAs to target E6 and E7, additional restoration of p53 and pRb was not induced, but processing of caspases and PARP was increased compared with wogonin treatment alone. Together, our findings demonstrated that wogonin effectively promotes apoptosis by downregulating E6 and E7 expressions and promoting intrinsic apoptosis in human cervical cancer cells.

The biflavonoid amentoflavone induces apoptosis via suppressing E7 expression, cell cycle arrest at sub-G1 phase, and mitochondria-emanated intrinsic pathways in human cervical cancer cells.

Amentoflavone, a biflavonoid from Selaginella tamariscina, is known to possess several bioactivities such as antitumor, anti-inflammatory, and antifungal effects. However, the mechanism of the anticancer effects of amentoflavone on human cervical cancer cells has not been studied in detail. In this study, we demonstrated that amentoflavone induces apoptosis in SiHa and CaSki cervical cancer cells by suppressing human papillomavirus protein E7 expression. The cyclins and tumor suppressors were modulated by amentoflavone in SiHa and CaSki human cervical cancer cells: cyclin and hyperphosphorylated retinoblastoma (p-pRb) were down-regulated, whereas cyclin-dependent kinase inhibitors and p53 were enhanced. Amentoflavone up-regulated peroxisome proliferator-activated receptor γ (PPARγ) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression levels while inhibiting E7-mediated cyclooxygenase-2 (COX-2)/interleukin-32 (IL-32) expressions were downregulated, and Akt phosphorlylation was decreased in an amentoflavone-induced apoptotic process, suggesting that amentoflavone may be a PPARγ activator. Additionally, the expression of the anti-apoptotic factor Bcl-2 was decreased, whereas that of the well-known apoptotic factor Bax was increased, thereby releasing cytochrome c into cytosol in amentoflavone-treated cervical cancer cells. Furthermore, amentoflavone treatment led to the activation of caspase-3 and -9 and proteolytic cleavage of poly(ADP-ribose) polymerase. The expression level of the extrinsic death receptor Fas (CD95) was not altered by amentoflavone treatment. When these findings are taken together, the biflavonoid amentoflavone activates PPARγ/PTEN expressions and induces apoptosis via suppressing E7 expression, cell cycle arrest at sub-G1 phase, and mitochondria-emanated intrinsic pathways in SiHa and CaSki human cervical cancer cells. These findings suggest that amentoflavone has potential for development as a therapeutic agent for human cervical cancer.

Cellular factor YY1 downregulates the human papillomavirus 16 E6/E7 promoter, P97, in vivo and in vitro from a negative element overlapping the transcription-initiation site.

Cellular factors that bind to cis sequences in the human papillomavirus 16 (HPV-16) upstream regulatory region (URR) positively and negatively regulate the viral E6 and E7 oncogene promoter, P97. DNase I footprinting has revealed the binding of cellular proteins to two previously undetected cis elements overlapping and 3' of the transcription-initiation site of the P97 promoter. Mutations within homologous motifs found in both of these cis elements abolished their negative function in vivo and the binding of the same cellular complex in vitro. This factor was identified as YY1 by complex mobility and binding specificity in comparison with vaccinia virus-expressed, purified recombinant YY1 protein and by antigenic reactivity with YY1 antisera. Cis mutations in the 'initiator' YY1 site activated the P97 promoter in vivo and in vitro. P97 was also activated threefold in vitro by depletion of endogenous YY1 with wild-type, but not mutant, YY1 oligonucleotides from the IgH kappa E3' enhancer. Furthermore, increasing concentrations of exogenous, purified recombinant YY1 repressed wild-type P97 transcript levels by up to threefold, but did not influence the P97 promoter mutated in the 'initiator' YY1 site. Thus, the promoter-proximal YY1 site was not necessary for correct transcription initiation at the P97 promoter, but was found to be required for downregulation of P97 transcription in vivo and in vitro. In contrast to other viral and cellular promoters, where YY1 is thought to function as a positive transcription-'initiator' factor, HPV-16 P97 transcription is downregulated by YY1 from a critical motif overlapping the transcription start site.

Loss of p53 induces epidermal growth factor receptor promoter activity in normal human keratinocytes.

Overexpression of the epidermal growth factor receptor (EGFR) in human papillomavirus type 16-immortalized human keratinocytes (HKc) is caused by the viral oncoprotein E6, which targets p53 for degradation. We have previously observed that expression of p53 RNAi in normal HKc is associated with an increase in EGFR mRNA and protein. We now report that p53 RNAi induces EGFR promoter activity up to approximately 10-fold in normal HKc, and this effect does not require intact p53 binding sites on the EGFR promoter. Exogenous wild-type p53 inhibits the EGFR promoter at low levels, and activates it at higher concentrations. Yin Yang 1 (YY1), which negatively regulates p53, induces EGFR promoter activity, and this effect is augmented by p53 RNAi. Intact p53 binding sites on the EGFR promoter are not required for activation by YY1. In addition, Sp1 and YY1 synergistically induce the EGFR promoter in normal HKc, indicating that Sp1 may recruit YY1 as a co-activator. Wild-type p53 suppressed Sp1- and YY1-mediated induction of the EGFR promoter. We conclude that acute loss of p53 in normal HKc induces EGFR expression by a mechanism that involves YY1 and Sp1 and does not require p53 binding to the EGFR promoter.