Caveolin-1 and lipids: Association and their dualism in oncogenic regulation.

Caveolin-1 (Cav-1) is a structural protein of caveolae that functions as a molecular organizer for different cellular functions including endocytosis and cellular signaling. Cancer cells take advantage of the physical position of Cav-1, as it can communicate with extracellular matrix, help to organize growth factor receptors, redistribute cholesterol and glycosphingolipids, and finally transduce signals within the cells for oncogenesis. Recent studies emphasize the exceeding involvement of Cav-1 with different lipid bodies and in altering the metabolism, especially lipid metabolism. However, the association of Cav-1 with different lipid bodies like lipid rafts, lipid droplets, cholesterols, sphingolipids, and fatty acids is remarkably dynamic. The lipid-Cav-1 alliance plays a dual role in carcinogenesis. Both cancer progression and regression are modified and affected by the type of lipid molecule's association with Cav-1. Accordingly, this Cav-1-lipid cooperation exemplifies a cancer-type-specific treatment strategy for a better prognosis of the disease. In this review, we first present Cav-1 as an oncogenic molecule and its communication via lipid raft. We discussed the involvement of Cav-1 with lipid droplets, Cholesterol, sphingolipids, gangliosides, and ceramides. Further, we describe the Cav-1-mediated altered Fatty acid metabolism in cancer and the strategic therapeutic approaches toward Cav-1 targeting.

Lymphatic vasculature in ovarian cancer.

Ovarian cancer (OVCA) is the second most common gynecological cancer and one of the leading causes of cancer related mortality among women. Recent studies suggest that among ovarian cancer patients at least 70% of the cases experience the involvement of lymph nodes and metastases through lymphatic vascular network. However, the impact of lymphatic system in the growth, spread and the evolution of ovarian cancer, its contribution towards the landscape of ovarian tissue resident immune cells and their metabolic responses is still a major knowledge gap. In this review first we present the epidemiological aspect of the OVCA, the lymphatic architecture of the ovary, we discuss the role of lymphatic circulation in regulation of ovarian tumor microenvironment, metabolic basis of the upregulation of lymphangiogenesis which is often observed during progression of ovarian metastasis and ascites development. Further we describe the implication of several mediators which influence both lymphatic vasculature as well as ovarian tumor microenvironment and conclude with several therapeutic strategies for targeting lymphatic vasculature in ovarian cancer progression in present day.

Anticancer Properties of Kaempferol on Cellular Signaling Pathways.

Polyhydroxy compounds are secondary metabolites that are ubiquitous in plants of higher genera. They possess therapeutic properties against a wide spectrum of diseases, including cancers, neurodegenerative disorders, atherosclerosis, as well as cardiovascular disease. The phytochemical flavonol (a type of flavonoid) kaempferol (KMP) (3,5,7-trihydroxy-2-(4-hydroxyphenyl)- 4Hchromen-4-one) is abundant in cruciferous vegetables, including broccoli, kale, spinach, and watercress, as well as in herbs like dill, chives, and tarragon. KMP is predominantly hydrophobic in nature due to its diphenylpropane structure (a characteristic feature of flavonoids). Recent findings have indicated the promise of applying KMP in disease prevention due to its potential antioxidant, antimutagenic, antifungal, and antiviral activities. In the literature, there is evidence that KMP exerts its anticancer effects by modulating critical elements in cellular signal transduction pathways linked to apoptosis, inflammation, angiogenesis, and metastasis in cancer cells without affecting the viability of normal cells. It has been shown that KMP triggers cancer cell death by several mechanisms, including cell cycle arrest, caspase activation, metabolic alteration, and impacting human telomerase reverse-transcriptase gene expression. This review is aimed at providing critical insights into the influence of KMP on the intracellular cascades that regulate metabolism and signaling in breast, ovarian, and cervical cancer cells.

Nuclear localization of histamine receptor 2 in primary human lymphatic endothelial cells.

Histamine exerts its physiological functions through its four receptor subtypes. In this work, we report the subcellular localization of histamine receptor 2 (H2R), a G protein-coupled receptor (GPCR), which is expressed in a wide variety of cell and tissue types. A growing number of GPCRs have been shown to be localized in the nucleus and contribute toward transcriptional regulation. In this study, for the first time, we demonstrate the nuclear localization of H2R in lymphatic endothelial cells. In the presence of its ligand, we show significant upregulation of H2R nuclear translocation kinetics. Using fluorescently tagged histamine, we explored H2R-histamine binding interaction, which exhibits a critical role in this translocation event. Altogether, our results highlight the previously unrecognized nuclear localization pattern of H2R. At the same time, H2R as a GPCR imparts many unresolved questions, such as the functional relevance of this localization, and whether H2R can contribute directly to transcriptional regulation and can affect lymphatic specific gene expression. H2R blockers are commonly used medications that recently have shown significant side effects. Therefore, it is imperative to understand the precise molecular mechanism of H2R biology. In this aspect, our present data shed new light on the unexplored H2R signaling mechanisms. This article has an associated First Person interview with the first author of the paper.

Multi-component redox system for selective and potent antineoplastic activity towards ovarian cancer cells.

Ovarian cancer is the deadliest gynecological cancer which rarely causes symptoms, and goes undetected until reaching the advanced stage of drug-resistant metastases. The cationic porphyrin meso-tetra(4-N-methylpyridyl)porphine (TMPyP) is a well-known photosensitizer (PS) used in photodyamic therapy (PDT) for curing cancer due to its strong affinity for DNA and high yield of reactive oxygen species (ROS) upon light activation. The practicality to irradiate tumor cells alone in the physiological system being slim (due to the close proximity of healthy cells and tumors), we looked for a variation in the PDT using a mixture of TMPyP with 1,5-dihydroxynapthalene (DHN) and Fe(III) ions at a mole ratio of 1:20:17 (drug combo) respectively in aqueous solution. The drug combo needs no photoactivation in H2O2 rich environment (mimicking the microenvironment of cancer/tumor), where it generates È®H and juglone, the latter being a known potent anticancer agent. In vitro studies of the drug combo in drug resistant and sensitive ovarian cancer cell lines showed drastic growth inhibition and cell death compared to normal epithelial cells. The drug combo provides an effective and non-invasive alternative to conventional PDT, exploiting the cytosolic carcinogenic H2O2 to produce an efficient anticancer treatment. The unique action of cancer-specific cytotoxicity arises from the redox chemistry involving activation of Fe(III) as the oxidizing agent to generate juglone, which utilizes the cytosolic ROS in cancer cells against itself.

Targeting LRRC15 Inhibits Metastatic Dissemination of Ovarian Cancer.

Dissemination of ovarian cancer cells can lead to inoperable metastatic lesions in the bowel and omentum that cause patient death. Here we show that LRRC15, a type-I 15-leucine-rich repeat-containing membrane protein, highly overexpressed in ovarian cancer bowel metastases compared with matched primary tumors and acts as a potent promoter of omental metastasis. Complementary models of ovarian cancer demonstrated that LRRC15 expression leads to inhibition of anoikis-induced cell death and promotes adhesion and invasion through matrices that mimic omentum. Mechanistically, LRRC15 interacted with ß1-integrin to stimulate activation of focal adhesion kinase (FAK) signaling. As a therapeutic proof of concept, targeting LRRC15 with the specific antibody-drug conjugate ABBV-085 in both early and late metastatic ovarian cancer cell line xenograft models prevented metastatic dissemination, and these results were corroborated in metastatic patient-derived ovarian cancer xenograft models. Furthermore, treatment of 3D-spheroid cultures of LRRC15-positive patient-derived ascites with ABBV-085 reduced cell viability. Overall, these data uncover a role for LRRC15 in promoting ovarian cancer metastasis and suggest a novel and promising therapy to target ovarian cancer metastases.Significance: This study identifies that LRRC15 activates ß1-integrin/FAK signaling to promote ovarian cancer metastasis and shows that the LRRC15-targeted antibody-drug conjugate ABBV-085 suppresses ovarian cancer metastasis in preclinical models.

Health disparity and COVID-19-A retrospective analysis.

BACKGROUND AND AIMS: According to the World Health Organization (WHO), more than 75.7 million confirmed cases of coronavirus disease 2019 (COVID-19), a global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have been reported so far. Researchers are working relentlessly to find effective solutions to this catastrophe, using genomic sequence-based investigation, immunological analysis, and more. The role of health disparity has also emerged as an intriguing factor that made a huge impact on the lives of people. METHODS: We analyzed various factors that triggered the health disparity in the United States of America along with the rate of COVID-19 morbidity and mortality. Furthermore, we have also focused on the State of Mississippi, which is suffering from an extreme health disparity. Data have been obtained from publicly available data sources including, Center for Disease Control and Prevention and Mississippi State Department of Health. Correlation analysis of the dataset has been performed using R software. RESULTS: Our analysis suggested that the COVID-19 infection rate per 100 000 people is directly correlated with the increasing number of the African American population in the United States. We have found a strong correlation between the obesity and the COVID-19 cases as well. All the counties in Mississippi demonstrate a strong correlation between a higher number of African American population to COVID-19 cases and obesity. Our data also indicate that a higher number of African American populations are facing socioeconomic disadvantages, which enhance their chances of becoming vulnerable to pre-existing ailments such as obesity, type-2 diabetes, and cardiovascular diseases. CONCLUSION: We proposed a possible explanation of increased COVID-19 infectivity in the African American population in the United States. This work has highlighted the intriguing factors that increased the health disparity at the time of the COVID-19 pandemic.

Group III phospholipase A2 downregulation attenuated survival and metastasis in ovarian cancer and promotes chemo-sensitization.

BACKGROUND: Aberrant lipogenicity and deregulated autophagy are common in most advanced human cancer and therapeutic strategies to exploit these pathways are currently under consideration. Group III Phospholipase A2 (sPLA2-III/PLA2G3), an atypical secretory PLA2, is recognized as a regulator of lipid metabolism associated with oncogenesis. Though recent studies reveal that high PLA2G3 expression significantly correlates with poor prognosis in several cancers, however, role of PLA2G3 in ovarian cancer (OC) pathogenesis is still undetermined. METHODS: CRISPR-Cas9 and shRNA mediated knockout and knockdown of PLA2G3 in OC cells were used to evaluate lipid droplet (LD) biogenesis by confocal and Transmission electron microscopy analysis, and the cell viability and sensitization of the cells to platinum-mediated cytotoxicity by MTT assay. Regulation of primary ciliation by PLA2G3 downregulation both genetically and by metabolic inhibitor PFK-158 induced autophagy was assessed by immunofluorescence-based confocal analysis and immunoblot. Transient transfection with GFP-RFP-LC3B and confocal analysis was used to assess the autophagic flux in OC cells. PLA2G3 knockout OVCAR5 xenograft in combination with carboplatin on tumor growth and metastasis was assessed in vivo. Efficacy of PFK158 alone and with platinum drugs was determined in patient-derived primary ascites cultures expressing PLA2G3 by MTT assay and immunoblot analysis. RESULTS: Downregulation of PLA2G3 in OVCAR8 and 5 cells inhibited LD biogenesis, decreased growth and sensitized cells to platinum drug mediated cytotoxicity in vitro and in in vivo OVCAR5 xenograft. PLA2G3 knockdown in HeyA8MDR-resistant cells showed sensitivity to carboplatin treatment. We found that both PFK158 inhibitor-mediated and genetic downregulation of PLA2G3 resulted in increased number of percent ciliated cells and inhibited cancer progression. Mechanistically, we found that PFK158-induced autophagy targeted PLA2G3 to restore primary cilia in OC cells. Of clinical relevance, PFK158 also induces percent ciliated cells in human-derived primary ascites cells and reduces cell viability with sensitization to chemotherapy. CONCLUSIONS: Taken together, our study for the first time emphasizes the role of PLA2G3 in regulating the OC metastasis. This study further suggests the therapeutic potential of targeting phospholipases and/or restoration of PC for future OC treatment and the critical role of PLA2G3 in regulating ciliary function by coordinating interface between lipogenesis and metastasis.

Transepithelial Anti-Neuroblastoma Response to Kale among Four Vegetable Juices Using In Vitro Model Co-Culture System.

Juicing vegetables is thought to be an anticancer treatment. Support exists for a rank order of anticancer greens (kale > dandelion > lettuce > spinach) based on degrees of bioavailability of different phytochemicals, also offset by some noxious molecules (i.e., calcium-oxalate). We developed a new in vitro transepithelial anti-neuroblastoma model system. The juices were diluted as predicted once in the small intestine. They were applied to apical Caco-2Bbe1 cells atop dividing SH-SY5Y neuroblastoma cells, and changes in transepithelial electrical resistance (TEER) and cell growth were considered with juice spectroscopies. Studied first in monoculture, kale and dandelion were the most cytostatic juices on SH-SY5Ys, lettuce showed no effect, and high (4.2%) spinach was cytotoxic. In co-culture, high (4.2%) kale was quickest (three days) to inhibit neuroblastoma growth. By five days, dandelion and kale were equally robust. Lettuce showed small anti-proliferative effects at five days and spinach remained cytotoxic. Spinach's cytotoxicity corresponded with major infrared bands indicative of oxalate. Kale juice uniquely induced reactive oxygen species and S-phase cell cycle arrest in SH-SY5Y. The superiority of kale and dandelion was also apparent on the epithelium, because raising TEER levels is considered healthy. Kale's unique features corresponded with a major fluorescent peak that co-eluted with kaempferol during high performance liquid chromatography. Because the anticancer rank order was upheld, the model appears validated for screening anticancer juices.

Sulfated glycolipid PG545 induces endoplasmic reticulum stress and augments autophagic flux by enhancing anticancer chemotherapy efficacy in endometrial cancer.

The sulfated glycolipid PG545 shows promising antitumor activity in various cancers. This study was conducted to explore the effects and the mechanism of PG545 action in endometrial cancer (EC). PG545 exhibited strong synergy as assessed by the Chou-Talalay-Method in vitro when combined with cisplatin, or paclitaxel in both type I (Hec1B) and type II (ARK2) EC cell lines. While PG545 showed antitumor activity as monotherapy, a combination of PG545 with paclitaxel and cisplatin was highly effective in reducing the tumor burden and significantly prolonged survival of both Hec1B and ARK2 xenograft bearing mice. Mechanistically, PG545 elicits ER stress as an early response with resultant induction of autophagy. Our data demonstrated an increase in pERK, Bip/Grp78, IRE1α, Calnexin and CHOP/GADD153 within 6-24 hrs of PG545 treatment in EC cells. In parallel, PG545 also blocked FGF2 and HB-EGF mediated signaling in EC cells. Moreover, melatonin-mediated ER stress inhibition reduced PG545-mediated autophagy and PG545 in combination with cisplatin further heightened this stress response. Collectively these data indicate that PG545 exhibits strong synergistic effects with chemotherapeutics in vitro and showed promising antitumor activity in vivo. Our preclinical data indicates that in future studies PG545 can be a useful adjunct to chemotherapy in endometrial cancer.

Studying nanoscale structural alterations in cancer cells to evaluate ovarian cancer drug treatment, using transmission electron microscopy imaging.

Understanding nanoscale structural changes can provide information about the physical state of cells/tissues. It has now been shown that increases in nanoscale structural alterations are associated with the progress of carcinogenesis in most cancer cases, including early carcinogenesis. Anti-cancerous therapies are designed to inhibit the growth of cancer cells; however, it is challenging to detect the efficacy of such drugs in the early stages of treatment. A unique method of assessing the impact of anti-cancerous drugs on cancerous cells/tissues is to probe the nanoscale structural alterations. In this paper, we study the effect of different anti-cancerous drugs on ovarian tumorigenic cells, using their nanoscale structural alterations as a biomarker. Transmission electron microscopy (TEM) imaging on thin cell sections is performed to obtain their nanoscale structures. The degree of nanoscale structural alterations of tumorigenic cells and anti-cancerous drug treated tumorigenic cells are quantified by using the recently developed inverse participation ratio (IPR) technique. Results show an increase in the degree of nanoscale fluctuations in tumorigenic cells relative to non-tumorigenic cells; then a near-reversal of the degree of fluctuation in tumorigenic cells to that in non-tumorigenic cells, following anti-cancerous drug treatment. These results support that the effect of anti-cancerous drugs in cancer treatment can be quantified by using the degree of nanoscale fluctuations in the cells via TEM imaging. Potential applications of the technique for cancer treatment are also discussed.

PFKFB3 inhibition reprograms malignant pleural mesothelioma to nutrient stress-induced macropinocytosis and ER stress as independent binary adaptive responses.

The metabolic signatures of cancer cells are often associated with elevated glycolysis. Pharmacological (PFK158 treatment) and genetic inhibition of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), a critical control point in the glycolytic pathway, decreases glucose uptake, ATP production, and lactate dehydrogenase activity and arrests malignant pleural mesothelioma (MPM) cells in the G0/G1 phase to induce cell death. To overcome this nutrient stress, inhibition of PFKFB3 activity led to an escalation in endoplasmic reticulum (ER) activity and aggravated ER stress mostly by upregulating BiP and GADD153 expression and activation of the endocytic Rac1-Rab5-Rab7 pathway resulting in a unique form of cell death called "methuosis" in both the sarcomatoid (H28) and epithelioid (EMMeso) cells. Transmission electron microscopy (TEM) analysis showed the formation of nascent macropinocytotic vesicles, which rapidly coalesced to form large vacuoles with compromised lysosomal function. Both immunofluorescence microscopy and co-immunoprecipitation analyses revealed that upon PFKFB3 inhibition, two crucial biomolecules of each pathway, Rac1 and Calnexin interact with each other. Finally, PFK158 alone and in combination with carboplatin-inhibited tumorigenesis of EMMeso xenografts in vivo. Since most cancer cells exhibit an increased glycolytic rate, these results provide evidence for PFK158, in combination with standard chemotherapy, may have a potential in the treatment of MPM.

Genes associated with bowel metastases in ovarian cancer.

OBJECTIVE: This study is designed to identify genes and pathways that could promote metastasis to the bowel in high-grade serous ovarian cancer (OC) and evaluate their associations with clinical outcomes. METHODS: We performed RNA sequencing of OC primary tumors (PTs) and their corresponding bowel metastases (n = 21 discovery set; n = 18 replication set). Differentially expressed genes (DEGs) were those expressed at least 2-fold higher in bowel metastases (BMets) than PTs in at least 30% of patients (P < .05) with no increased expression in paired benign bowel tissue and were validated with quantitative reverse transcription PCR. Using an independent OC cohort (n = 333), associations between DEGs in PTs and surgical and clinical outcomes were performed. Immunohistochemistry and mouse xenograft studies were performed to confirm the role of LRRC15 in promoting metastasis. RESULTS: Among 27 DEGs in the discovery set, 21 were confirmed in the replication set: SFRP2, Col11A1, LRRC15, ADAM12, ADAMTS12, MFAP5, LUM, PLPP4, FAP, POSTN, GRP, MMP11, MMP13, C1QTNF3, EPYC, DIO2, KCNA1, NETO1, NTM, MYH13, and PVALB. Higher expression of more than half of the genes in the PT was associated with an increased requirement for bowel resection at primary surgery and an inability to achieve complete cytoreduction. Increased expression of LRRC15 in BMets was confirmed by immunohistochemistry and knockdown of LRRC15 significantly inhibited tumor progression in mice. CONCLUSIONS: We identified 21 genes that are overexpressed in bowel metastases among patients with OC. Our findings will help select potential molecular targets for the prevention and treatment of malignant bowel obstruction in OC.

A Child with Partial Trisomy 4 (q26 - qterminal) Resulting from Paternally Inherited Translocation (4:18) Associated with Multiple Congenital Anomalies and Death.

Parental balanced reciprocal translocations can result in partial aneuploidy in the offspring due to unbalanced meiotic segregation during gametogenesis. Herein, we report the phenotypic and cytogenetic characterization in a 9-day-old male child with partial trisomy of chromosome 4. Karyotyping of the proband and parents was performed along with multicolor fluorescence in situ hybridization (mFISH) of paternal chromosomes. Conventional cytogenetic analysis by karyotyping showed 47,XY,der(18),t(4;18)(q26;q22),+4 in proband, and the paternal karyotype was found as 47,XY,der(18),t(4;18)(q26;q22). mFISH analysis on paternal chromosomal preparations confirmed both region and origin of the balanced translocation. In this study, karyotyping helped us to identify both numerical and structural anomalies in the proband, and mFISH helped us to confirm our cytogenetic findings. Therefore, cytogenetic screening of both partners is recommended before pregnancy to rule out or confirm the presence of any numerical or structural anomaly in one, both, or none of the partners.

Therapeutic targeting of PFKFB3 with a novel glycolytic inhibitor PFK158 promotes lipophagy and chemosensitivity in gynecologic cancers.

Metabolic alterations are increasingly recognized as important novel anti-cancer targets. Among several regulators of metabolic alterations, fructose 2,6 bisphosphate (F2,6BP) is a critical glycolytic regulator. Inhibition of the active form of PFKFB3ser461 using a novel inhibitor, PFK158 resulted in reduced glucose uptake, ATP production, lactate release as well as induction of apoptosis in gynecologic cancer cells. Moreover, we found that PFK158 synergizes with carboplatin (CBPt) and paclitaxel (PTX) in the chemoresistant cell lines, C13 and HeyA8MDR but not in their chemosensitive counterparts, OV2008 and HeyA8, respectively. We determined that PFK158-induced autophagic flux leads to lipophagy resulting in the downregulation of cPLA2, a lipid droplet (LD) associated protein. Immunofluorescence and co-immunoprecipitation revealed colocalization of p62/SQSTM1 with cPLA2 in HeyA8MDR cells uncovering a novel pathway for the breakdown of LDs promoted by PFK158. Interestingly, treating the cells with the autophagic inhibitor bafilomycin A reversed the PFK158-mediated synergy and lipophagy in chemoresistant cells. Finally, in a highly metastatic PTX-resistant in vivo ovarian mouse model, a combination of PFK158 with CBPt significantly reduced tumor weight and ascites and reduced LDs in tumor tissue as seen by immunofluorescence and transmission electron microscopy compared to untreated mice. Since the majority of cancer patients will eventually recur and develop chemoresistance, our results suggest that PFK158 in combination with standard chemotherapy may have a direct clinical role in the treatment of recurrent cancer.

Arachidonic Acid Induces the Migration of MDA-MB-231 Cells by Activating Raft-associated Leukotriene B4 Receptors.

BACKGROUND: The migration of tumor cells is critical in spreading cancers through the lymphatic nodes and circulatory systems. Although arachidonic acid (AA) and its soluble metabolites have been shown to induce the migration of breast and colon cancer cells, the mechanism by which it induces such migration has not been fully understood. OBJECTIVE: The effect of AA on migratory responses of the MDA-MB-231 cell line (a triple-negative breast cancer cell) was examined and compared with MCF-7 (estrogen-receptor positive) breast cancer cells to elucidate the mechanism of AA-induced migration. METHODS: Migrations of breast cancer cells were examined with the help of wound-healing assays. AA-induced eicosanoid synthesis was monitored by RP-HPLC. Cellular localizations of lipoxygenase and lipid rafts were assessed by immunoblot and confocal microscopy. RESULTS: AA treatment stimulated the synthesis of leukotriene B4 (LTB4) and HETE-8, but lowered the levels of prostaglandin E2 (PGE2), prostaglandin D2 (PGD2), and HETE-5 in MDA-MB-231 cells. Further analysis indicated that AA increased the expression of 5-lipoxygenase (5-LOX) in this cell line and inhibiting its expression by small molecule inhibitors lowered the production of LTB4 and reduced migration. In contrast, MCF-7 cells did not show any appreciable changes in eicosanoid synthesis, 5-LOX expression, or cellular migration. CONCLUSION: Our results suggest that AA treatment activates the BLT1 receptor (present in membrane microdomains) and stimulates the synthesis of LTB4 production, which is likely to be associated with the migration of MDA-MB-231 cells.

Quinacrine upregulates p21/p27 independent of p53 through autophagy-mediated downregulation of p62-Skp2 axis in ovarian cancer.

We have previously shown that the anti-malarial compound Quinacrine (QC) inhibits ovarian cancer (OC) growth by modulating autophagy. In the present study we extended these studies to identify the molecular pathways regulated by QC to promote apoptosis independent of p53 status in OC. QC exhibited strong anti-cancer properties in OC cell lines in contrast to other anti-malarial autophagy inhibiting drugs. QC treatment selectively upregulated cell cycle inhibitor p21, and downregulated F box protein Skp2 and p62/SQSTM1 expression independent of p53 status. Genetic downregulation of key autophagy protein ATG5 abolished QC-mediated effects on both cell cycle protein p21/Skp2 as well as autophagic cargo protein p62. Furthermore, genetic silencing of p62/SQSTM1 resulted in increased sensitivity to QC-mediated apoptosis, downregulated Skp2 mRNA and increased accumulation of p21 expression. Likewise, genetic knockdown of Skp2 resulted in the upregulation of p21 and p27 and increased sensitivity of OC cells to QC treatment. In contrast, transient overexpression of exogenous p62-HA plasmid rescued the QC-mediated Skp2 downregulation indicating the positive regulation of Skp2 by p62. Collectively, these data indicate that QC-mediated effects on cell cycle proteins p21/Skp2is autophagy-dependent and p53-independent in high grade serious OC cells.

Quinacrine in endometrial cancer: Repurposing an old antimalarial drug.

OBJECTIVE: Generate preclinical data on the effect of quinacrine (QC) in inhibiting tumorigenesis in endometrial cancer (EC) in vitro and explore its role as an adjunct to standard chemotherapy in an EC mouse model. METHODS: Five different EC cell lines (Ishikawa, Hec-1B, KLE, ARK-2, and SPEC-2) representing different histologies, grades of EC, sensitivity to cisplatin and p53 status were used for the in vitro studies. MTT and colony formation assays were used to examine QC's ability to inhibit cell viability in vitro. The Chou-Talalay methodology was used to examine synergism between QC and cisplatin, carboplatin or paclitaxel. A cisplatin-resistant EC subcutaneous mouse model (Hec-1B) was used to examine QC's role as maintenance therapy. RESULTS: QC exhibited strong synergism in vitro when combined with cisplatin, carboplatin or paclitaxel with the highest level of synergism in the most chemo-resistant cell line. Neither QC monotherapy nor carboplatin/paclitaxel significantly delayed tumor growth in xenografts. Combination treatment (QC plus carboplatin/paclitaxel) significantly augmented the antiproliferative ability of these agents and was associated with a 14-week survival prolongation compared to carboplatin/paclitaxel. Maintenance with QC resulted in further delay in tumor progression and survival prolongation compared to carboplatin/paclitaxel. QC was not associated with weight loss and the yellow skin discoloration noted during treatment was reversible upon discontinuation. CONCLUSIONS: QC exhibited significant antitumor activity against EC in vitro and was successful as maintenance therapy in chemo-resistant EC mouse xenografts. This preclinical data suggest that QC may be an important adjunct to standard chemotherapy for patients with chemo-resistant EC.

Loss of HSulf-1: The Missing Link between Autophagy and Lipid Droplets in Ovarian Cancer.

Defective autophagy and deranged metabolic pathways are common in cancer; pharmacologic targeting of these two pathways could provide a viable therapeutic option. However, how these pathways are regulated by limited availability of growth factors is still unknown. Our study shows that HSulf-1 (endosulfatase), a known tumor suppressor which attenuates heparin sulfate binding growth factor signaling, also regulates interplay between autophagy and lipogenesis. Silencing of HSulf-1 in OV202 and TOV2223 cells (ovarian cancer cell lines) resulted in increased lipid droplets (LDs), reduced autophagic vacuoles (AVs) and less LC3B puncta. In contrast, HSulf-1 proficient cells exhibit more AVs and reduced LDs. Increased LDs in HSulf-1 depleted cells was associated with increased ERK mediated cPLA2S505 phosphorylation. Conversely, HSulf-1 expression in SKOV3 cells reduced the number of LDs and increased the number of AVs compared to vector controls. Furthermore, pharmacological (AACOCF3) and ShRNA mediated downregulation of cPLA2 resulted in reduced LDs, and increased autophagy. Finally, in vivo experiment using OV202 Sh1 derived xenograft show that AACOCF3 treatment effectively attenuated tumor growth and LD biogenesis. Collectively, these results show a reciprocal regulation of autophagy and lipid biogenesis by HSulf-1 in ovarian cancer.

Quinacrine promotes autophagic cell death and chemosensitivity in ovarian cancer and attenuates tumor growth.

A promising new strategy for cancer therapy is to target the autophagic pathway. In the current study, we demonstrate that the antimalarial drug Quinacrine (QC) reduces cell viability and promotes chemotherapy-induced cell death in an autophagy-dependent manner more extensively in chemoresistant cells compared to their isogenic chemosensitive control cells as quantified by the Chou-Talalay methodology. Our preliminary data, in vitro and in vivo, indicate that QC induces autophagy by downregulating p62/SQSTM1 to sensitize chemoresistant cells to autophagic- and caspase-mediated cell death in a p53-independent manner. QC promotes autophagosome accumulation and enhances autophagic flux by clearance of p62 in chemoresistant ovarain cancer (OvCa) cell lines to a greater extent compared to their chemosensitive controls. Notably, p62 levels were elevated in chemoresistant OvCa cell lines and knockdown of p62 in these cells resulted in a greater response to QC treatment. Bafilomycin A, an autophagy inhibitor, restored p62 levels and reversed QC-mediated cell death and thus chemosensitization. Importantly, our in vivo data shows that QC alone and in combination with carboplatin suppresses tumor growth and ascites in the highly chemoresistant HeyA8MDR OvCa model compared to carboplatin treatment alone. Collectively, our preclinical data suggest that QC in combination with carboplatin can be an effective treatment for patients with chemoresistant OvCa.