Diane-35 and Metformin Induce Autophagy and Apoptosis in Polycystic Ovary Syndrome Women with Early-Stage Endometrial Carcinoma

Objective: Women with polycystic ovary syndrome (PCOS) are at increased risk ofendometrial carcinoma (EC). Previous studies indicated that the combined therapy of Diane-35 and metformin significantly suppresses disease progression in PCOS patients with early EC; however, the mechanisms remain unclear. Methods: An established murine model of PCOS with early EC, clinical specimens, and human EC cells was used in this study. The levels of protein and mRNA were measured with Western blotting and RT-PCR, respectively. Cell proliferation was determined with MTT, colony formation, and flow cytometry. Proteins were analyzed with immunofluorescence and immunohistochemistry. Results: Diane-35 and metformin significantly inhibited proliferative activity and promoted apoptosis in EC cells. Additionally, cell autophagy was induced by the combined therapy. Quantitive PCR revealed that Diane-35 and metformin decreased androgen receptor (AR) expression but elevated GLUT4 expression. AR was found to repress GLUT4 expression by binding to the promoter of GLUT4. Moreover, the combined treatment mediated the onset of cellular autophagy by regulating the mTORC pathway via the suppression of IGF-1 and inhibited the development of EC by the activation of the PI3K/mTORC pathway. Conclusion: The results and previous clinical evidence support the use of Diane-35 and metformin combination therapy for patients with PCOS and early EC.

A mathematical model of p62-ubiquitin aggregates in autophagy

Aggregation of ubiquitinated cargo by oligomers of the protein p62 is an important preparatory step in cellular autophagy. In this work a mathematical model for the dynamics of these heterogeneous aggregates in the form of a system of ordinary differential equations is derived and analyzed. Three different parameter regimes are identified, where either aggregates are unstable, or their size saturates at a finite value, or their size grows indefinitely as long as free particles are abundant. The boundaries of these regimes as well as the finite size in the second case can be computed explicitly. The growth in the third case (quadratic in time) can also be made explicit by formal asymptotic methods. In the absence of rigorous results the dynamic stability of these structures has been investigated by numerical simulations. A comparison with recent experimental results permits a partial parametrization of the model.

Antiproliferative and cytotoxic activity of Geraniaceae plant extracts against five tumor cell lines

Aim: To determine the antiproliferative and cytotoxic activities of Geranium and Erodium species against human cancer and noncancer cell lines, respectively. Methods: Twenty-one species of Geranium and Erodium were extracted and screened against cancerous and noncancerous human cell lines. Results: In a dose-response manner, G. glaberrimum, G. asphodeloides, E. brandianum and E. leucanthum were able, with variable potency, to inhibit cellular proliferation. Except for E. brandianum, all extracts induced cellular autophagy in tumor cells with similar levels to that of rapamycin; but, only E. brandianum induced cellular apoptosis, likely through Bcl2 and BAX protein expressions. Discussion: This is the first study to report the potential antiproliferative effects of ethanol extracts of several Geraniaceae species.

Long Non-coding RNA ENST00000453774.1 Confers an Inhibitory Effect on Renal Fibrosis by Inhibiting miR-324-3p to Promote NRG1 Expression

Progressive or chronic renal diseases arise from a process of destructive renal fibrosis. Therefore, the molecular basis of renal fibrosis has attracted increasing attention. In this investigation, we set out to elucidate the potential interaction among long non-coding RNA ENST00000453774.1 (lncRNA 74.1), microRNA-324-3p (miR-324-3p), and NRG1, and to investigate their roles in the context of cellular autophagy and renal fibrosis. We collected 30 renal fibrosis tissue samples for analysis. In other studies, HK-2 cells were stimulated with TGF-β1 to induce a cell model of renal fibrosis, followed by alteration on the expression of lncRNA 74.1, miR-324-3p, or NRG1, or by the addition of AKT activator SC79 in the HK-2 cells. The expression levels of lncRNA 74.1, miR-324-3p, NRG1, autophagy-related proteins (ATG5, ATG7, LC3II/I, and P62), and the corresponding fibrosis markers (Collagen I, Fibronectin, and α-SMA) were subsequently determined using various assay methods. In addition, the proportion of LC3 positive cells and number of autophagosomes were recorded. Results revealed that lncRNA 74.1 and NRG1 were poorly expressed and miR-324-3p was highly expressed in renal fibrosis tissues and modeled cells. LncRNA 74.1 could bind to miR-324-3p, which led to upregulated NRG1 expression and inhibition of the PI3K/AKT signaling pathway. Meanwhile, overexpression of lncRNA 74.1 or down-regulation of miR-324-3p increased the levels of ATG5, ATG7, LC3II, and LC3I, and decreased levels of P62, Collagen I, Fibronectin, and α-SMA, accompanied by elevated proportions of LC3 positive cells and autophagosomes. Findings concur in showing that lncRNA 74.1 could induce cellular autophagy and alleviate renal fibrosis by regulating the miR-324-3p-mediated NRG1/PI3K/AKT axis. This axis may thus present a potential molecular target in renal fibrosis treatment.

Research Progress on Fumonisin B1 Contamination and Toxicity: A Review

Fumonisin B1 (FB1), belonging to the member of fumonisins, is one of the most toxic mycotoxins produced mainly by Fusarium proliferatum and Fusarium verticillioide. FB1 has caused extensive contamination worldwide, mainly in corn, rice, wheat, and their products, while it also poses a health risk and is toxic to animals and human. It has been shown to cause oxidative stress, endoplasmic reticulum stress, cellular autophagy, and apoptosis. This review focuses on the current stage of FB1 contamination, its toxic effects of acute toxicity, immunotoxicity, organ toxicity, and reproductive toxicity on animals and humans. The potential toxic mechanisms of FB1 are discussed. One of the main aims of the work is to provide a reliable reference strategy for understanding the occurrence and toxicity of FB1.

Effective Targeting of Raf-1 and Its Associated Autophagy by Novel Extracted Peptide for Treating Breast Cancer Cells

Breast cancer is one of the most common causes of death in women worldwide and has harmful influence on their psychological state during therapy. Multikinase inhibitors have become effective drugs for treating a variety of cancer diseases such as breast cancer. A purified short peptide (H-P) was isolated from the natural honey and tested for its potential regulatory role in breast cancer cells compared with the effectiveness of the anticancer drug, Sorafenib (SOR), using MCF-7, EFM-19, and MCF-10A cell lines. Furthermore, we investigated the direct connection between Raf-1 activation and cellular autophagy as potential targets of SOR and H-P extract using RNA interference. Interestingly, the treatment with H-P showed competitive regulation of phosphorylated Raf-1, MEK1/2, and matched autophagy-related LC3B without any detectable toxic effects in the non-tumorigenic epithelial cells. Unlike SOR, the regulation of Raf-1 protein and autophagic machinery by the novel H-P extract showed neglected levels of the released proinflammatory cytokine. This regulation of cytokine secretion by H-P resulted in decreasing the expression level of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) in treated cells. Moreover, the transfection of MCF-7 cells with small interference RNA (siRNA) antagonist Raf-1 expression markedly reduced the expression of LC3B, while it increased the expression of NF-kB1 and NF-kB2, indicating the potential cross-link between Raf-1, autophagy, and NF-kB effector. Collectively, these findings suggest that H-P-mediated Raf-1, MEK1/2, LC3B, and NF-kB provide a novel and efficacious multikinase inhibitor for treating breast cancer without detectable cytotoxic effects.

Interaction of Poliovirus Capsid Proteins with the Cellular Autophagy Pathway

The capsid precursor P1 constitutes the N-terminal part of the enterovirus polyprotein. It is processed into VP0, VP3, and VP1 by the viral proteases, and VP0 is cleaved autocatalytically into VP4 and VP2. We observed that poliovirus VP0 is recognized by an antibody against a cellular autophagy protein, LC3A. The LC3A-like epitope overlapped the VP4/VP2 cleavage site. Individually expressed VP0-EGFP and P1 strongly colocalized with a marker of selective autophagy, p62/SQSTM1. To assess the role of capsid proteins in autophagy development we infected different cells with poliovirus or encapsidated polio replicon coding for only the replication proteins. We analyzed the processing of LC3B and p62/SQSTM1, markers of the initiation and completion of the autophagy pathway and investigated the association of the viral antigens with these autophagy proteins in infected cells. We observed cell-type-specific development of autophagy upon infection and found that only the virion signal strongly colocalized with p62/SQSTM1 early in infection. Collectively, our data suggest that activation of autophagy is not required for replication, and that capsid proteins contain determinants targeting them to p62/SQSTM1-dependent sequestration. Such a strategy may control the level of capsid proteins so that viral RNAs are not removed from the replication/translation pool prematurely.

ORF3a-Mediated Incomplete Autophagy Facilitates Severe Acute Respiratory Syndrome Coronavirus-2 Replication

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the causative agent for the coronavirus disease 2019 (COVID-19) pandemic and there is an urgent need to understand the cellular response to SARS-CoV-2 infection. Beclin 1 is an essential scaffold autophagy protein that forms two distinct subcomplexes with modulators Atg14 and UVRAG, responsible for autophagosome formation and maturation, respectively. In the present study, we found that SARS-CoV-2 infection triggers an incomplete autophagy response, elevated autophagosome formation but impaired autophagosome maturation, and declined autophagy by genetic knockout of essential autophagic genes reduces SARS-CoV-2 replication efficiency. By screening 26 viral proteins of SARS-CoV-2, we demonstrated that expression of ORF3a alone is sufficient to induce incomplete autophagy. Mechanistically, SARS-CoV-2 ORF3a interacts with autophagy regulator UVRAG to facilitate PI3KC3-C1 (Beclin-1-Vps34-Atg14) but selectively inhibit PI3KC3-C2 (Beclin-1-Vps34-UVRAG). Interestingly, although SARS-CoV ORF3a shares 72.7% amino acid identity with the SARS-CoV-2 ORF3a, the former had no effect on cellular autophagy response. Thus, our findings provide the mechanistic evidence of possible takeover of host autophagy machinery by ORF3a to facilitate SARS-CoV-2 replication and raise the possibility of targeting the autophagic pathway for the treatment of COVID-19.