Natural Flavonoid Chrysin Cuts HPV16 E6 Oncoprotein and Reshapes microRNAs in Cervical Cancer Cells

A naturally occurring plant flavonoid may be able to weaken one of the central molecular engines of cervical cancer, according to a new laboratory study published in Medical Oncology. Researchers report that chrysin, a compound found in propolis, honey, and several edible plants, reduced the viability of HPV16-positive CaSki cervical cancer cells while sparing normal human dermal fibroblasts, and that this selective toxicity was accompanied by a significant drop in the levels of the viral E6 oncoprotein and by pronounced changes in the expression of five cancer-related microRNAs. The work, led by Mahdis Gholami Fashkhami, Hadi Habibollahi, and Seyedeh Tooba Shafighi of the Department of Biology at Islamic Azad University’s Rasht Branch in Iran, adds to a growing body of evidence that phytochemicals can interfere with the molecular machinery that high-risk human papillomaviruses use to drive cells toward malignancy.

The biological rationale behind the study rests on the well-established role of HPV16 in cervical carcinogenesis. Persistent infection with high-risk HPV types is the major cause of cervical cancer worldwide, and the viral oncoproteins E6 and E7 are the principal effectors of that transformation. E6 promotes the ubiquitin-mediated degradation of the p53 tumor suppressor, disabling a cell’s primary safeguard against DNA damage and uncontrolled proliferation, while E7 disrupts the retinoblastoma protein pathway. Beyond these canonical functions, the viral oncoproteins also reshape the landscape of microRNA expression in infected cells. MicroRNAs are short, non-coding RNA molecules that regulate gene expression after transcription by binding target messenger RNAs and suppressing their translation or promoting their degradation. Because a single microRNA can influence dozens of targets, shifts in microRNA profiles can ripple across entire signaling networks, and viral manipulation of these regulators is increasingly recognized as a hallmark of HPV-associated cancers.

To test whether chrysin could counteract these effects, the team treated CaSki cells, a cervical cancer cell line that carries integrated HPV16 DNA, alongside normal human dermal fibroblasts as a non-cancerous comparison. The cells were exposed to chrysin across a concentration range of 31.5 to 500 micrograms per milliliter for two exposure periods of 24 and 48 hours. Cell viability was measured using the MTT assay, a colorimetric method in which metabolically active cells convert a yellow tetrazolium salt into a purple formazan product, allowing the proportion of surviving cells to be quantified. The researchers then quantified the expression of five selected microRNAs, miR-34a, miR-218, miR-20a, miR-195, and miR-23b, by quantitative reverse transcription polymerase chain reaction, calculating relative expression with the 2 to the power of negative delta delta Ct method. Levels of the HPV16 E6 protein were assessed by Western blotting, which separates proteins by molecular weight and detects the target with specific antibodies.

The viability results were striking for their selectivity. Chrysin reduced the survival of CaSki cells in a dose- and time-dependent manner, but it showed minimal toxicity toward the normal dermal fibroblasts at the same concentrations, yielding a high selectivity index. This therapeutic window matters because many candidate anticancer compounds fail precisely at this hurdle, killing tumor cells and healthy tissue alike. A high selectivity index suggests that the compound’s cytotoxic mechanisms preferentially target vulnerabilities present in the cancer cells, such as the altered survival signaling and metabolic rewiring characteristic of HPV-transformed lines, rather than general cellular processes shared by all dividing cells.

The microRNA findings provided the study’s most detailed molecular picture. After chrysin treatment, miR-34a expression rose approximately 10.05-fold compared with untreated controls, a change that reached a statistical significance level of P less than 0.0001. miR-218 increased 5.64-fold with P less than 0.01, and miR-20a showed a more moderate 1.7-fold increase with P less than 0.05. In contrast, miR-195 expression fell to 0.18-fold of control levels with P less than 0.01, while miR-23b showed no statistically significant change. Each of these microRNAs has documented connections to cancer biology. miR-34a is a transcriptional target of p53 and one of the best-characterized tumor-suppressive microRNAs, and previous work has shown that the HPV E6 oncoprotein interrupts its expression, effectively severing a p53-dependent regulatory arm. The restoration of miR-34a after chrysin treatment is therefore consistent with the concurrent reduction in E6 protein that the team observed.

miR-218, the second most strongly induced microRNA in the study, functions as a tumor suppressor in cervical cancer, where it has been shown to inhibit epithelial-to-mesenchymal transition, migration, and invasion by targeting genes such as SFMBT1 and DCUN1D1 and by acting within the SLIT/ROBO signaling pathway. Its fivefold increase after chrysin exposure suggests that the flavonoid may reinforce anti-metastatic regulatory circuits in addition to affecting viral oncoprotein abundance. The moderate rise in miR-20a is more nuanced, because members of the miR-17-92 cluster to which it belongs can behave as oncomiRs in some contexts while showing context-dependent effects in others. The decrease in miR-195, which has been reported to play roles in gynecological cancers with both tumor-suppressive and proliferative implications depending on setting, likewise underscores that microRNA responses to phytochemicals are not uniformly one-directional and require interpretation against specific cellular backgrounds.

Perhaps the most consequential measurement was the Western blot result for the viral oncoprotein itself. Chrysin-treated CaSki cells showed a significant reduction in HPV16 E6 protein levels, with treated samples measuring 0.64 relative to untreated controls, a difference significant at P less than 0.001. Because E6 is the viral factor responsible for targeting p53 for degradation, lowering E6 abundance could, in principle, allow p53-dependent tumor-suppressive programs to partially recover. The observed tenfold induction of miR-34a, a canonical p53-responsive microRNA, fits this interpretation, although the study’s design establishes association rather than a fully mapped causal chain. The authors are careful to frame the findings accordingly, noting that the concurrent modulation of E6 and several cancer-related microRNAs indicates a link but that the underlying pathways remain to be clarified.

The study also situates itself within a broader research effort on chrysin, a flavone with the chemical name 5,7-dihydroxyflavone. Prior investigations have documented apoptotic effects of chrysin in various human cancer cell lines, selective antiproliferative and antimigratory activity across a panel of cervical cancer-derived lines, inhibition of HeLa cell propagation through attenuation of cell survival signaling, and induction of both intrinsic and extrinsic apoptosis in ovarian cancer cells delivered via chrysin-loaded micelles. Chrysin has also been reported to alter microRNA expression in gastric cancer cells, suggesting that modulation of post-transcriptional regulation may be a recurring feature of its anticancer activity. At the same time, the compound faces a familiar obstacle for polyphenolic phytochemicals: poor aqueous solubility and limited bioavailability, which have motivated formulation strategies such as nanoparticle and micellar delivery systems in recent preclinical work.

For the cervical cancer field specifically, the results contribute to an emerging interest in natural products as sources of multi-target agents against HPV-driven disease. Because E6 and E7 are usually maintained in cervical tumors even as other viral genes are lost, they represent attractive therapeutic targets, and compounds that reduce E6 protein levels could complement existing prevention and treatment strategies, from vaccination and screening to surgery, radiotherapy, and chemotherapy. The microRNA dimension adds a further layer of potential value, since circulating miR-34a, miR-218, and related species have been proposed as early, minimally invasive biomarkers of cervical carcinogenesis, and microRNA profiles could in principle serve as pharmacodynamic readouts of a compound’s activity.

The authors and outside observers alike emphasize the preliminary nature of the work. All experiments were conducted in a single HPV16-positive cell line with one normal comparator, over exposure windows of 24 and 48 hours, and the study did not generate or analyze external datasets. Whether the E6 reduction results from effects on viral gene transcription, on E6 messenger RNA stability, on protein translation, or on protein degradation pathways, and whether the microRNA changes drive the cytotoxicity or merely accompany it, are questions the current design cannot answer. The researchers state that further studies in additional cellular models and mechanistic systems are required to clarify the underlying pathways. Translational development would also need to confront chrysin’s bioavailability limitations and establish efficacy and safety in animal models before any clinical relevance could be assessed. For now, the study offers a carefully documented snapshot of a common dietary flavonoid simultaneously touching a viral oncoprotein and a network of cancer-related microRNAs in cervical cancer cells, a combination that justifies closer mechanistic scrutiny.

Subject of Research: Effects of the flavonoid chrysin on HPV16 E6 oncoprotein levels and cancer-related microRNA expression in cervical cancer cells

Article Title: Chrysin treatment is associated with altered microrna expression and reduced levels of HPV16 E6 protein in caski cervical cancer cells

Article References: Gholami Fashkhami, M., Habibollahi, H., & Shafighi, S. T. (2026). Chrysin treatment is associated with altered microrna expression and reduced levels of HPV16 E6 protein in caski cervical cancer cells. Medical Oncology, 43(10), Article 257. https://doi.org/10.1007/s12032-026-03381-9

Image Credits: AI Generated

DOI: 10.1007/s12032-026-03381-9

Keywords: chrysin, cervical cancer, HPV16, E6 oncoprotein, microRNA, CaSki cells, flavonoid, miR-34a, miR-218, phytochemical, cell viability, Medical Oncology