Dysregulation of Human Hepatic Drug Transporters by Proinflammatory Cytokines.

Proinflammatory cytokines, elevated during inflammation caused by infection and/or autoimmune disorders, result in reduced clearance of drugs eliminated primarily by cytochrome P450 enzymes (CYPs). However, the effect of cytokines on hepatic drug transporter expression or activity has not been well-studied. Here, using plated human hepatocytes (PHHs; n = 3 lots), we investigated the effect of interleukin (IL)-6, IL-1ß, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ), on the mRNA expression and activity of hepatic drug transporters. PHHs were incubated for 72 hours at their pathophysiologically relevant plasma concentrations, both individually (0.01, 0.1, 1, 10 ng/ml) or as a cocktail (i.e., when each was combined at 0.1 or 1 ng/ml). Following cytokine cocktail exposure (1 ng/ml), significant downregulation of mRNA expression of organic anion transporting polypeptide 1B1 (OATP1B1), OATP1B3, sodium/taurocholate cotransporting polypeptide (NTCP), breast cancer resistance protein (BCRP), P-glycoprotein (P-gp), multidrug and toxin extrusion protein 1, multidrug resistance proteins (MRP) 2, 3, and 4 was observed. While the mRNA expression of organic anion transporter (OAT) 2 and organic cation transporter (OCT) 1 was downregulated in two lots, it was upregulated in one lot. In agreement (mostly), the 1 ng/ml cytokine cocktail reduced OATP1B1/3, OATP2B1, OAT2, OCT1, and NTCP activity by 75%, 44%, 82%, 47%, and 80%, respectively. Interestingly, upregulation of OAT2 and OCT1 mRNA in one donor did not translate into the same directional change in activity. Although significant interlot variability was observed, in general, the above effects, using individual cytokines, could be attributed to IL-1ß, TNF-α, and IFN-γ. SIGNIFICANCE STATEMENT: To date, this is the first comprehensive study to investigate the effect of four major proinflammatory cytokines, both individually and as a cocktail, on the mRNA expression and activity of human hepatic drug transporters. The data obtained can be used in the future to predict transporter-mediated drug clearance changes during inflammation through physiologically based pharmacokinetic modeling and simulation.

Dysregulation of the mRNA Expression of Human Renal Drug Transporters by Proinflammatory Cytokines in Primary Human Proximal Tubular Epithelial Cells.

Proinflammatory cytokines, which are elevated during inflammation or infections, can affect drug pharmacokinetics (PK) due to the altered expression or activity of drug transporters and/or metabolizing enzymes. To date, such studies have focused on the effect of cytokines on the activity and/or mRNA expression of hepatic transporters and drug-metabolizing enzymes. However, many antibiotics and antivirals used to treat infections are cleared by renal transporters, including the basal organic cation transporter 2 (OCT2), organic anion transporters 1 and 3 (OAT1 and 3), the apical multidrug and toxin extrusion proteins 1 and 2-K (MATE1/2-K), and multidrug resistance-associated protein 2 and 4 (MRP2/4). Here, we determined the concentration-dependent effect of interleukin-6 (IL-6), IL-1ß, tumor necrosis factor (TNF)-α, and interferon-γ (IFN-γ) on the mRNA expression of human renal transporters in freshly isolated primary human renal proximal tubular epithelial cells (PTECs, n = 3-5). PTECs were exposed to either a cocktail of cytokines, each at 0.01, 0.1, 1, or 10 ng/mL or individually at the same concentrations. Exposure to the cytokine cocktail for 48 h was found to significantly downregulate the mRNA expression, in a concentration-dependent manner, of OCT2, the organic anion transporting polypeptides 4C1 (OATP4C1), OAT4, MATE2-K, P-glycoprotein (P-gp), and MRP2 and upregulate the mRNA expression of the organic cation/carnitine transporter 1 (OCTN1) and MRP3. OAT1 and OAT3 also appeared to be significantly downregulated but only at 0.1 and 10 ng/mL, respectively, without a clear concentration-dependent trend. Among the cytokines, IL-1ß appeared to be the most potent at down- and upregulating the mRNA expression of the transporters. Taken together, our results demonstrate for the first time that proinflammatory cytokines transcriptionally dysregulate renal drug transporters in PTECs. Such dysregulation could potentially translate into changes in transporter protein abundance or activity and alter renal transporter-mediated drug PK during inflammation or infections.

Linoleic acid exhibits anti-proliferative and anti-invasive activities in endometrial cancer cells and a transgenic model of endometrial cancer.

Emerging evidence has provided considerable insights into the integral function of reprogramming fatty acid metabolism in the carcinogenesis and progression of endometrial cancer. Linoleic acid, an essential fatty acid with the highest consumption in the Western diet regimen, has shown pro-tumorigenic or anti-tumorigenic effects on tumor cell growth and invasion in multiple types of cancer. However, the biological role of linoleic acid in endometrial cancer remains unclear. In the present study, we aimed to investigate the functional impact of linoleic acid on cell proliferation, invasion, and tumor growth in endometrial cancer cells and in a transgenic mouse model of endometrial cancer. The results showed that Linoleic acid significantly inhibited the proliferation of endometrial cancer cells in a dose-dependent manner. The treatment of HEC-1A and KLE cells with linoleic acid effectively increased intracellular reactive oxygen species (ROS) production, decreased mitochondrial membrane potential, caused cell cycle G1 arrest, and induced intrinsic and extrinsic apoptosis pathways. The anti-invasive ability of linoleic acid was found to be associated with the epithelial-mesenchymal transition process in both cell lines, including the decreased expression of N-cadherin, snail, and vimentin. Furthermore, treatment of Lkb1fl/flp53fl/fl transgenic mice with linoleic acid for four weeks significantly reduced the growth of endometrial tumors and decreased the expression of VEGF, vimentin, Ki67, and cyclin D1 in tumor tissues. Our findings demonstrate that linoleic acid exhibits anti-proliferative and anti-invasive activities in endometrial cancer cell lines and the Lkb1fl/flp53fl/fl mouse model of endometrial cancer, thus providing a pre-clinical basis for future dietary interventions with linoleic acid in endometrial cancer.

Palmitic Acid Exerts Anti-Tumorigenic Activities by Modulating Cellular Stress and Lipid Droplet Formation in Endometrial Cancer.

Epidemiological and clinical evidence have extensively documented the role of obesity in the development of endometrial cancer. However, the effect of fatty acids on cell growth in endometrial cancer has not been widely studied. Here, we reported that palmitic acid significantly inhibited cell proliferation of endometrial cancer cells and primary cultures of endometrial cancer and reduced tumor growth in a transgenic mouse model of endometrial cancer, in parallel with increased cellular stress and apoptosis and decreased cellular adhesion and invasion. Inhibition of cellular stress by N-acetyl-L-cysteine effectively reversed the effects of palmitic acid on cell proliferation, apoptosis, and invasive capacity in endometrial cancer cells. Palmitic acid increased the intracellular formation of lipid droplets in a time- and dose-dependent manner. Depletion of lipid droplets by blocking DGAT1 and DGAT2 effectively increased the ability of palmitic acid to inhibit cell proliferation and induce cleaved caspase 3 activity. Collectively, this study provides new insight into the effect of palmitic acid on cell proliferation and invasion and the formation of lipid droplets that may have potential clinical relevance in the treatment of obesity-driven endometrial cancer.

A novel dopamine receptor D2 antagonist (ONC206) potentiates the effects of olaparib in endometrial cancer.

Poly ADP-ribose polymerase (PARP) inhibitors are effective therapies for cancer patients with homologous recombination (HR) deficient tumors. The imipridone ONC206 is an orally bioavailable dopamine receptor D2 antagonist and mitochondrial protease ClpP agonist that has anti-tumorigenic effects in endometrial cancer via induction of apoptosis, activation of the integrated stress response and modulation of PI3K/AKT signaling. Both PARP inhibitors and imipridones are being evaluated in endometrial cancer clinical trials but have yet to be explored in combination. In this manuscript, we evaluated the effects of the PARP inhibitor olaparib in combination with ONC206 in human endometrioid endometrial cancer cell lines and in a genetically engineered mouse model of endometrial cancer. Our results showed that simultaneous exposure of endometrial cancer cells to olaparib and ONC206 resulted in synergistic anti-proliferative effects and increased cellular stress and apoptosis in both cell lines, compared to either drug alone. The combination treatment also decreased expression of the anti-apoptotic protein Bcl-2 and reduced phosphorylation of AKT and S6, with greater effects compared to either drug alone. In the transgenic model of endometrial cancer, the combination of olaparib and ONC206 resulted in a more significant reduction in tumor weight in obese and lean mice compared to ONC206 alone or olaparib alone, together with a considerably decreased Ki-67 and enhanced H2AX expression in obese and lean mice. These results suggest that this novel dual therapy may be worthy of further exploration in clinical trials.

Ipatasertib exhibits anti­tumorigenic effects and enhances sensitivity to paclitaxel in endometrial cancer in vitro and in vivo.

Endometrial cancer is the most common gynecologic cancer and one of the only cancers for which incidence and mortality is steadily increasing. Although curable with surgery in the early stages, endometrial cancer presents a significant clinical challenge in the metastatic and recurrent setting with few novel treatment strategies emerging in the past fifty years. Ipatasertib (IPAT) is an orally bioavailable pan­AKT inhibitor, which targets all three AKT isoforms and has demonstrated anti­tumor activity in pre­clinical models, with clinical trials emerging for many cancer types. In the present study, the MTT assay was employed to evaluate the therapeutic efficacy of IPAT or IPAT in combination with paclitaxel (PTX) in endometrial cancer cell lines and primary cultures of endometrial cancer. The effect of IPAT and PTX on the growth of endometrial tumors was evaluated in a transgenic mouse model of endometrial cancer. Apoptosis was assessed using cleaved caspase assays and cellular stress was assessed using ROS, JC1 and tetramethylrhodamine ethyl ester assays. The protein expression levels of markers of apoptosis and cellular stress, and DNA damage were evaluated using western blotting and immunohistochemistry. IPAT significantly inhibited cell proliferation, caused cell cycle G1 phase arrest, and induced cellular stress and mitochondrial apoptosis in a dose dependent manner in human endometrial cancer cell lines. Combined treatment with low doses of IPAT and PTX led to synergistic inhibition of cell proliferation and induction of cleaved caspase 3 activity in the human endometrial cancer cell lines and the primary cultures. Furthermore, IPAT effectively reduced tumor growth, accompanied by decreased protein expression levels of Ki67 and phosphorylation of S6 in the Lkb1fl/flp53fl/fl mouse model of endometrioid endometrial cancer. The combination of IPAT and PTX resulted in increased expression of phosphorylated­H2AX and KIF14, markers of DNA damage and microtubule dysfunction respectively, as compared with IPAT alone, PTX alone or placebo­treated mice. The results of the present study provide a biological rationale to evaluate IPAT and the combination of IPAT and PTX in future clinical trials for endometrial cancer.

Oleic Acid Exhibits Anti-Proliferative and Anti-Invasive Activities via the PTEN/AKT/mTOR Pathway in Endometrial Cancer.

Reprogramming of fatty acid metabolism promotes cell growth and metastasis through a variety of processes that stimulate signaling molecules, energy storage, and membrane biosynthesis in endometrial cancer. Oleic acid is one of the most important monounsaturated fatty acids in the human body, which appears to have both pro- and anti-tumorigenic activities in various pre-clinical models. In this study, we evaluated the potential anti-tumor effects of oleic acid in endometrial cancer cells and the LKB1fl/flp53fl/fl mouse model of endometrial cancer. Oleic acid increased lipogenesis, inhibited cell proliferation, caused cell cycle G1 arrest, induced cellular stress and apoptosis, and suppressed invasion in endometrial cancer cells. Targeting of diacylglycerol acyltransferases 1 and 2 effectively increased the cytotoxicity of oleic acid. Moreover, oleic acid significantly increased the expression of wild-type PTEN, and knockdown of PTEN by shRNA partially reversed the anti-proliferative and anti-invasive effects of oleic acid. Inhibition of the AKT/mTOR pathway by ipatasertib effectively increased the anti-tumor activity of oleic acid in endometrial cancer cells. Oleic acid treatment (10 mg/kg, daily, oral) for four weeks significantly inhibited tumor growth by 52.1% in the LKB1fl/flp53fl/fl mice. Our findings demonstrated that oleic acid exhibited anti-tumorigenic activities, dependent on the PTEN/AKT/mTOR signaling pathway, in endometrial cancer.

Asparagus officinalis combined with paclitaxel exhibited synergistic anti-tumor activity in paclitaxel-sensitive and -resistant ovarian cancer cells.

PURPOSE: Although paclitaxel is a promising first-line chemotherapeutic drug for ovarian cancer, acquired resistance to paclitaxel is one of the leading causes of treatment failure, limiting its clinical application. Asparagus officinalis has been shown to have anti-tumorigenic effects on cell growth, apoptosis, cellular stress and invasion of various types of cancer cells and has also been shown to synergize with paclitaxel to inhibit cell proliferation in ovarian cancer. METHODS: Human ovarian cancer cell lines MES and its PTX-resistant counterpart MES-TP cell lines were used and were treated with Asparagus officinalis and paclitaxel alone as well as in combination. Cell proliferation, cellular stress, invasion and DMA damage were investigated and the synergistic effect of a combined therapy analyzed. RESULTS: In this study, we found that Asparagus officinalis combined with low-dose paclitaxel synergistically inhibited cell proliferation, induced cellular stress and apoptosis and reduced cell invasion in paclitaxel-sensitive and -resistant ovarian cancer cell lines. The combined treatment effects were dependent on DNA damage pathways and suppressing microtubule dynamics, and the AKT/mTOR pathway and microtubule-associated proteins regulated the inhibitory effect through different mechanisms in paclitaxel-sensitive and -resistant cells. CONCLUSION: These findings suggest that the combination of Asparagus officinalis and paclitaxel have potential clinical implications for development as a novel ovarian cancer treatment strategy.

Contrastive Proposal Extension With LSTM Network for Weakly Supervised Object Detection.

Weakly supervised object detection (WSOD) has attracted more and more attention since it only uses image-level labels and can save huge annotation costs. Most of the WSOD methods use Multiple Instance Learning (MIL) as their basic framework, which regard it as an instance classification problem. However, these methods based on MIL tend to only converge on the most discriminative regions of different instances, rather than their corresponding complete regions, that is, insufficient integrity. Inspired by the human habit of observing things, we propose a new method by comparing the initial proposals and the extended ones to optimize those initial proposals. Specifically, we propose one new strategy for WSOD by involving contrastive proposal extension (CPE), which consists of multiple directional contrastive proposal extensions (D-CPEs), and each D-CPE contains LSTM-based encoders and dual-stream decoders. Firstly, the boundary of initial proposals in MIL is extended to different positions according to well-designed sequential order. Then, the CPE compares the extended proposal and the initial one by extracting the feature semantics of them using the encoders, and calculates the integrity of the initial proposal to optimize its score. These contrastive contextual semantics will guide the basic WSOD to suppress bad proposals and improve the scores of good ones. In addition, a simple dual-stream network is designed as the decoder to constrain the temporal coding of LSTM and improve the performance of WSOD further. Experiments on PASCAL VOC 2007, VOC 2012 and MS-COCO datasets show that our method has achieved the state-of-the-art results.

Ipatasertib, an oral AKT inhibitor, inhibits cell proliferation and migration, and induces apoptosis in serous endometrial cancer.

Ipatasertib (IPAT) is an orally administered, selective protein kinase B (AKT) inhibitor with promising data in solid tumors in both pre-clinical studies and clinical trials. Given that the PI3K/AKT/mTOR pathway is frequently dysregulated in uterine serous carcinoma (USC), we aimed to explore the functional impact of IPAT on anti-tumorigenic activity in USC cell lines and primary cultures of USC. We found that IPAT significantly inhibited cell proliferation and colony formation in a dose-dependent manner in USC cells. Induction of cell cycle arrest and apoptosis was observed in IPAT-treated ARK1 and SPEC-2 cells. Treatment with IPAT resulted in reduced adhesion and invasion of both cell lines with a concomitant decrease in the expression of Snail, Slug, and N-Cadherin. Compared with single-drug treatment, the combination of IPAT and paclitaxel synergistically reduced cell proliferation and increased the activity of cleaved caspase 3 in both cell lines. Additionally, IPAT inhibited growth in four of five primary USC cultures, and three of five primary cultures also exhibited synergistic growth inhibition when paclitaxel and IPAT were combined. These results support that IPAT appears to be a promising targeted agent in the treatment of USC.

Asparagus officinalis Exhibits Anti-Tumorigenic and Anti-Metastatic Effects in Ovarian Cancer.

Ovarian cancer is one of the leading causes of female cancer death. Emerging evidence suggests that many dietary natural products have anti-tumorigenic activity, including that of asparagus officinalis. The current study aimed to assess the anti-tumorigenic and anti-metastatic effects of asparagus officinalis on serous ovarian cancer cell lines and a transgenic mouse model of high grade serous ovarian cancer. Asparagus officinalis decreased cellular viability, caused cell cycle G1 phase arrest and induced apoptosis in the OVCAR5 and SKOV3 cells. Induction of apoptosis and inhibition of cell proliferation was rescued by the pan-caspase inhibitor, Z-VAD-FMK, implying that its cytotoxic effects were mainly dependent on caspase pathways. Asparagus officinalis increased levels of ROS and decreased mitochondrial membrane potential with corresponding increases in PERK, Bip, Calnexin PDI and ATF4 in both cell lines. Treatment with asparagus officinalis also reduced ability of adhesion and invasion through epithelial-mesenchymal transition and reduction of VEGF expression. The combination of Asparagus officinalis with paclitaxel had synergistic anti-proliferative activity. Furthermore, Asparagus officinalis significantly inhibited tumor growth and reduced serum VEGF in a genetically engineered mouse model of ovarian cancer under obese and lean conditions, accompanied with a decrease in the expression of Ki67, VEGF and phosphorylated S6, and in an increase in phosphorylation of AMPK in the ovarian tumor tissues. Overall, our data provide a pre-clinical rationale for asparagus officinalis in the prevention and treatment of ovarian cancer as a novel natural product.

Targeting dopamine receptor D2 as a novel therapeutic strategy in endometrial cancer.

BACKGROUND: ONC201 is a dopamine receptor D2 (DRD2) antagonist that inhibits tumor growth in preclinical models through ClpP activation to induce integrated stress response pathway and mitochondrial events related to inhibition of cell growth, which is being explored in clinical trials for solid tumors and hematological malignancies. In this study, we investigated the anti-tumorigenic effect of ONC201 in endometrial cancer cell lines and a genetically engineered mouse model of endometrial cancer. METHODS: Cell proliferation was assessed by MTT and colony formation assays. Cell cycle and apoptosis were evaluated by Cellometer. Invasion capacity was tested using adhesion, transwell and wound healing assays. LKB1fl/flp53fl/fl mouse model of endometrial cancer were fed a control low fat diet versus a high fat diet to mimic diet-induced obesity. Following tumor onset, mice were treated with placebo or ONC201. Metabolomics and lipidomics were used to identify the obesity-dependent effects of ONC201 in the mouse endometrial tumors. DRD2 expression was analyzed by immunohistochemistry in human endometrioid and serous carcinoma specimens. DRD2 mRNA expression from the Cancer Genome Atlas (TCGA) database was compared between the four molecular subtypes of endometrial cancer. RESULTS: Increasing DRD2 expression in endometrial cancer was significantly associated with grade, serous histology and stage, as well as worse progression free survival and overall survival. Higher expression of DRD2 mRNA was found for the Copy Number High (CNH) subtype when compared to the other subtypes. ONC201 inhibited cell proliferation, induced cell cycle G1 arrest, caused cellular stress and apoptosis and reduced invasion in endometrial cancer cells. Diet-induced obesity promoted endometrial tumor growth while ONC201 exhibited anti-tumorigenic efficacy in the obese and lean LKB1fl/fl/p53fl/fl mice. Metabolomic analysis demonstrated that ONC201 reversed the obesity-driven upregulation of lipid biosynthesis and reduced protein biosynthesis in obese and lean mice. CONCLUSION: ONC201 has anti-tumorigenic effects in endometrial cancer cells and a transgenic mouse model of endometrial cancer, and DRD2 expression was documented in both human serous and endometrioid endometrial cancer. These studies support DRD2 antagonism via ONC201 as a promising therapeutic strategy for endometrial cancer that has already demonstrated pharmacodynamic activity and clinical benefit in both serous and endometrioid endometrial cancer patients.