Pre-Treatment of Platinum Resistant Ovarian Cancer Cells with an MMP-9/MMP-2 Inhibitor Prior to Cisplatin Enhances Cytotoxicity as Determined by High Content Screening

Platinum resistance is a major cause of treatment failure in ovarian cancer. We previously identified matrix metalloproteinase 9 (MMP-9) as a potential therapeutic target of chemoresistant disease. A2780cis (cisplatin-resistant) and A2780 (cisplatin-sensitive) ovarian carcinoma cell lines were used. The cytotoxic effect of MMP-9/MMP-2 inhibitor, (2R)-2-[(4-Biphenylsulfonyl) amino]-3 phenylpropionic acid (C21H19NO4S) alone or in combination with cisplatin was determined using high content screening. Protein expression was examined using immunohistochemistry and ELISA. Co-incubation of cisplatin and an MMP-9/MMP-2 inhibitor, (2R)-2-[(4-Biphenylsulfonyl) amino]-3 phenylpropionic acid (C21H19NO4S) resulted in significantly greater cytotoxicity as compared to either treatment alone in a cisplatin resistant MMP-9 overexpressing cell line; A2780cis. In addition, pre-incubating with MMP-9i prior to cisplatin further enhances the cytotoxic effect. No significant difference was observed in MMP-9 protein in tissue but a trend towards increased MMP-9 was observed in recurrent serum. We propose that MMP-9/MMP-2i may be utilized in the treatment of recurrent/chemoresistant ovarian cancers that overexpress MMP-9 mRNA but its role in vivo remains to be evaluated.     

ARID1A Mutations and PI3K/AKT Pathway Alterations in Endometriosis and Endometriosis-Associated Ovarian Carcinomas

Endometriosis is a common gynecological disease affecting 6%–10% of women of reproductive age and is characterized by the presence of endometrial-like tissue in localizations outside of the uterine cavity as, e.g., endometriotic ovarian cysts. Mainly, two epithelial ovarian carcinoma subtypes, the ovarian clear cell carcinomas (OCCC) and the endometrioid ovarian carcinomas (EnOC), have been molecularly and epidemiologically linked to endometriosis. Mutations in the gene encoding the AT-rich interacting domain containing protein 1A (ARID1A) have been found to occur in high frequency in OCCC and EnOC. The majority of these mutations lead to a loss of expression of the ARID1A protein, which is a subunit of the SWI/SNF chromatin remodeling complex and considered as a bona fide tumor suppressor. ARID1A mutations frequently co-occur with mutations, leading to an activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, such as mutations in PIK3CA encoding the catalytic subunit, p110α, of PI3K. In combination with recent functional observations, these findings strongly suggest cooperating mechanisms between the two pathways. The occurrence of ARID1A mutations and alterations in the PI3K/AKT pathway in endometriosis and endometriosis-associated ovarian carcinomas, as well as the possible functional and clinical implications are discussed in this review.     

Inhibition of Glutamine Uptake Resensitizes Paclitaxel Resistance in SKOV3-TR Ovarian Cancer Cell via mTORC1/S6K Signaling Pathway

Ovarian cancer is a carcinoma that affects women and that has a high mortality rate. Overcoming paclitaxel resistance is important for clinical application. However, the effect of amino acid metabolism regulation on paclitaxel-resistant ovarian cancer is still unknown. In this study, the effect of an amino acid-deprived condition on paclitaxel resistance in paclitaxel-resistant SKOV3-TR cells was analyzed. We analyzed the cell viability of SKOV3-TR in culture conditions in which each of the 20 amino acids were deprived. As a result, the cell viability of the SKOV3-TR was significantly reduced in cultures deprived of arginine, glutamine, and lysine. Furthermore, we showed that the glutamine-deprived condition inhibited mTORC1/S6K signaling. The decreased cell viability and mTORC1/S6K signaling under glutamine-deprived conditions could be restored by glutamine and α-KG supplementation. Treatment with PF-4708671, a selective S6K inhibitor, and the selective glutamine transporter ASCT2 inhibitor V-9302 downregulated mTOR/S6K signaling and resensitized SKOV3-TR to paclitaxel. Immunoblotting showed the upregulation of Bcl-2 phosphorylation and a decrease in Mcl-1 expression in SKOV3-TR via the cotreatment of paclitaxel with PF-4708671 and V-9302. Collectively, this study demonstrates that the inhibition of glutamine uptake can resensitize SKOV3-TR to paclitaxel and represents a promising therapeutic target for overcoming paclitaxel resistance in ovarian cancer.     

Downregulation of MMP-9 Enhances the Anti-Migratory Effect of Cyclophosphamide in MDA-MB-231 and MCF-7 Breast Cancer Cell Lines

Metastasis is one of the most urgent issues in breast cancer patients. One of the factors necessary in the migration process is the remodeling of the extracellular matrix (ECM). Metalloproteinases (MMPs) can break down the elements of the ECM, which facilitates cell movement. Many highly aggressive tumors are characterized by high levels of MMPs. In the case of breast cancer, the association between MMP-9 and the migration potential and invasiveness of cells has been demonstrated. In addition, reports indicating increased migration of breast cancer cells after the administration of the commonly used cytostatic cyclophosphamide (CP) are particularly disturbing. Hence, our research aimed to assess the effect of CP treatment on MDA-MB-231 and MCF-7 cells and how this response is influenced by the downregulation of the MMP-9 level. The obtained results suggest that CP causes a decrease in the survival of breast cancer cells of various invasiveness, and the downregulation of MMP-9 enhances this effect, mainly by inducing apoptosis. Moreover, in the group of MMP-9 siRNA-transfected CP-treated cells, a more severe reduction in invasion and migration of cells of both lines was observed, as indicated by the migration and invasion transwell assays and Wound healing assay. Hence, we suggest that CP alone may not result in satisfactory therapeutic effects. On the other hand, the use of combination therapy targeting MMP-9, together with the CP, could improve the effectiveness of the treatment. Additionally, we confirmed a relationship between the levels of MMP-9 and cytokeratin 19 (CK19).     

BubR1 Acts as a Promoter in Cellular Motility of Human Oral Squamous Cancer Cells through Regulating MMP-2 and MMP-9

BubR1 is a critical component of spindle assembly checkpoint, ensuring proper chromatin segregation during mitosis. Recent studies showed that BubR1 was overexpressed in many cancer cells, including oral squamous cell carcinomas (OSCC). However, the effect of BubR1 on metastasis of OSCC remains unclear. This study aimed to unravel the role of BubR1 in the progression of OSCC and confirm the expression of BubR1 in a panel of malignant OSCC cell lines with different invasive abilities. The results of quantitative real-time PCR showed that the mRNA level of BubR1 was markedly increased in four OSCC cell lines, Ca9-22, HSC3, SCC9 and Cal-27 cells, compared to two normal cells, normal human oral keratinocytes (HOK) and human gingival fibroblasts (HGF). Moreover, the expression of BubR1 in these four OSCC cell lines was positively correlated with their motility. Immunofluorescence revealed that BubR1 was mostly localized in the cytosol of human gingival carcinoma Ca9-22 cells. BubR1 knockdown significantly decreased cellular invasion but slightly affect cellular proliferation on both Ca9-22 and Cal-27 cells. Consistently, the activities of metastasis-associated metalloproteinases MMP-2 and MMP-9 were attenuated in BubR1 knockdown Ca9-22 cells, suggesting the role of BubR1 in promotion of OSCC migration. Our present study defines an alternative pathway in promoting metastasis of OSCC cells, and the expression of BubR1 could be a prognostic index in OSCC patients.     

Elevated STAT3 Signaling-Mediated Upregulation of MMP-2/9 Confers Enhanced Invasion Ability in Multidrug-Resistant Breast Cancer Cells

The development of multidrug resistance greatly impedes effective cancer therapy. Recent advances in cancer research have demonstrated that acquisition of multidrug resistance by cancer cells is usually accompanied by enhanced cell invasiveness. Several lines of evidence indicated that cross activation of other signaling pathways during development of drug resistance may increase invasive potential of multidrug-resistant (MDR) cancer cells. However, the accurate mechanism of this process is largely undefined. In this study, to better understand the associated molecular pathways responsible for cancer progression induced by drug resistance, a MDR human breast cancer cell line SK-BR-3/EPR with P-glycoprotein overexpression was established using stepwise long-term exposure to increasing concentration of epirubicin. The SK-BR-3/EPR cell line exhibited decreased cell proliferative activity, but enhanced cell invasive capacity. We showed that the expression of metastasis-related matrix metalloproteinase (MMP)-2/9 was elevated in SK-BR-3/EPR cells. Moreover, SK-BR-3/EPR cells showed elevated activation of STAT3. Activation of STAT3 signaling is responsible for enhanced invasiveness of SK-BR-3/EPR cells through upregulation of MMP-2/9. STAT3 is a well-known oncogene and is frequently implicated in tumorigenesis and chemotherapeutic resistance. Our findings augment insight into the mechanism underlying the functional association between MDR and cancer invasiveness.     

A Novel L-Phenylalanine Dipeptide Inhibits the Growth and Metastasis of Prostate Cancer Cells via Targeting DUSP1 and TNFSF9

Prostate cancer (PCa) is a common malignant cancer of the urinary system. Drug therapy, chemotherapy, and radical prostatectomy are the primary treatment methods, but drug resistance and postoperative recurrence often occur. Therefore, seeking novel anti-tumor compounds with high efficiency and low toxicity from natural products can produce a new tumor treatment method. Matijin-Su [N-(N-benzoyl-L-phenylalanyl)-O-acetyl-L-phenylalanol, MTS] is a phenylalanine dipeptide monomer compound that is isolated from the Chinese ethnic medicine Matijin (Dichondra repens Forst.). Its derivatives exhibit various pharmacological activities, especially anti-tumor. Among them, the novel MTS derivative HXL131 has a significant inhibitory effect against prostate tumor growth and metastasis. This study is designed to investigate the effects of HXL131 on the growth and metastasis of human PCa cell lines PC3 and its molecular mechanism through in vitro experiments combined with proteomics, molecular docking, and gene silencing. The in vitro results showed that HXL131 concentration dependently inhibited PC3 cell proliferation, induced apoptosis, arrested cell cycle at the G2/M phase, and inhibited cell migration capacity. A proteomic analysis and a Western blot showed that HXL131 up-regulated the expression of proliferation, apoptosis, cell cycle, and migration-related proteins CYR61, TIMP1, SOD2, IL6, SERPINE2, DUSP1, TNFSF9, OSMR, TNFRSF10D, and TNFRSF12A. Molecular docking, a cellular thermal shift assay (CETSA), and gene silencing showed that HXL131 had a strong binding affinity with DUSP1 and TNFSF9, which are important target genes for inhibiting the growth and metastasis of PC3 cells. This study demonstrates that HXL131 exhibited excellent anti-prostate cancer activity and inhibited the growth and metastasis of prostate cancer cells by regulating the expression of DUSP1 and TNFSF9.     

Modulating the ERK1/2–MMP1 Axis through Corosolic Acid Inhibits Metastasis of Human Oral Squamous Cell Carcinoma Cells

Corosolic acid (CA; 2α-hydroxyursolic acid) is a natural pentacyclic triterpenoid with antioxidant, antitumour and antimetastatic activities against various tumour cells during tumourigenesis. However, CA’s antitumour effect and functional roles on human oral squamous cell carcinoma (OSCC) cells are utterly unknown. In this study, our results demonstrated that CA significantly exerted an inhibitory effect on matrix metalloproteinase (MMP)1 expression, cell migration and invasion without influencing cell growth or the cell cycle of human OSCC cells. The critical role of MMP1 was confirmed using the GEPIA database and showed that patients have a high expression of MMP1 and have a shorter overall survival rate, confirmed on the Kaplan–Meier curve assay. In the synergistic inhibitory analysis, CA and siMMP1 co-treatment showed a synergically inhibitory influence on MMP1 expression and invasion of human OSCC cells. The ERK1/2 pathway plays an essential role in mediating tumour progression. We found that CA significantly inhibits the phosphorylation of ERK1/2 dose-dependently. The ERK1/2 pathway played an essential role in the CA-mediated downregulation of MMP1 expression and in invasive motility in human OSCC cells. These findings first demonstrated the inhibitory effects of CA on OSCC cells’ progression through inhibition of the ERK1/2–MMP1 axis. Therefore, CA might represent a novel strategy for treating OSCC.     

Evodiamine Induces Apoptosis and Enhances TRAIL-Induced Apoptosis in Human Bladder Cancer Cells through mTOR/S6K1-Mediated Downregulation of Mcl-1

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), either alone or in combination with other anti-cancer agents, has been considered as a new strategy for anti-cancer therapy. In this study, we demonstrated that evodiamine, a quinolone alkaloid isolated from the fruit of Evodia fructus, induced apoptosis and enhanced TRAIL-induced apoptosis in human bladder cancer cells. To elucidate the underlying mechanism, we found that evodiamine significantly reduced the protein levels of Mcl-1 in 253J and T24 bladder cancer cells, and overexpression of this molecule attenuated the apoptosis induced by evodiamine alone, or in combination with TRAIL. Further experiments revealed that evodiamine did not affect the mRNA level, proteasomal degradation and protein stability of Mcl-1. On the other hand, evodiamine inhibited the mTOR/S6K1 pathway, which usually regulates protein translation; moreover, knockdown of S6K1 with small interfering RNA (siRNA) effectively reduced Mcl-1 levels, indicating evodiamine downregulates c-FLIP through inhibition of mTOR/S6K1 pathway. Taken together, our results indicate that evodiamine induces apoptosis and enhances TRAIL-induced apoptosis possibly through mTOR/S6K1-mediated downregulation of Mcl-1; furthermore, these findings provide a rationale for the combined application of evodiamine with TRAIL in the treatment of bladder cancer.     

Cardiac Protective Effect of Kirenol against Doxorubicin-Induced Cardiac Hypertrophy in H9c2 Cells through Nrf2 Signaling via PI3K/AKT Pathways

Kirenol (KRL) is a biologically active substance extracted from Herba Siegesbeckiae. This natural type of diterpenoid has been widely adopted for its important anti-inflammatory and anti-rheumatic properties. Despite several studies claiming the benefits of KRL, its cardiac effects have not yet been clarified. Cardiotoxicity remains a key concern associated with the long-term administration of doxorubicin (DOX). The generation of reactive oxygen species (ROS) causes oxidative stress, significantly contributing to DOX-induced cardiac damage. The purpose of the current study is to investigate the cardio-protective effects of KRL against apoptosis in H9c2 cells induced by DOX. The analysis of cellular apoptosis was performed using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining assay and measuring the modulation in the expression levels of proteins involved in apoptosis and Nrf2 signaling, the oxidative stress markers. Furthermore, Western blotting was used to determine cell survival. KRL treatment, with Nrf2 upregulation and activation, accompanied by activation of PI3K/AKT, could prevent the administration of DOX to induce cardiac oxidative stress, remodeling, and other effects. Additionally, the diterpenoid enhanced the activation of Bcl2 and Bcl-xL, while suppressing apoptosis marker proteins. As a result, KRL is considered a potential agent against hypertrophy resulting from cardiac deterioration. The study results show that KRL not only activates the IGF-IR-dependent p-PI3K/p-AKT and Nrf2 signaling pathway, but also suppresses caspase-dependent apoptosis.     

Pathogenic Role of the Sphingosine 1-Phosphate (S1P) Pathway in Common Gynecologic Disorders (GDs): A Possible Novel Therapeutic Target

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid, noteworthy for its involvement both in the modulation of various biological processes and in the development of many diseases. S1P signaling can be either pro or anti-inflammatory, and the sphingosine kinase (SphK)–S1P–S1P receptor (S1PR) axis is a factor in accelerating the growth of several cells, including endometriotic cells and fibrosis. Gynecologic disorders, including endometriosis, adenomyosis, and uterine fibroids are characterized by inflammation and fibrosis. S1P signaling and metabolism have been shown to be dysregulated in those disorders and they are likely implicated in their pathogenesis and pathophysiology. Enzymes responsible for inactivating S1P are the most affected by the dysregulation of S1P balanced levels, thus causing accumulation of sphingolipids within these cells and tissues. The present review highlights the past and latest evidence on the role played by the S1P pathways in common gynecologic disorders (GDs). Furthermore, it discusses potential future approaches in the regulation of this signaling pathway that could represent an innovative and promising therapeutical target, also for ovarian cancer treatment.     

Dihydromyricetin Inhibited Migration and Invasion by Reducing S100A4 Expression through ERK1/2/β-Catenin Pathway in Human Cervical Cancer Cell Lines

Cervical cancer has a poor prognosis and is the fourth most common cancer among women. Dihydromyricetin (DHM), a flavonoid compound, exhibits several pharmacological activities, including anticancer effects; however, the effects of DHM on cervical cancer have received insufficient research attention. This study examined the antitumor activity and underlying mechanisms of DHM on human cervical cancer. Our results indicated that DHM inhibits migration and invasion in HeLa and SiHa cell lines. Mechanistically, RNA sequencing analysis revealed that DHM suppressed S100A4 mRNA expression in HeLa cells. Moreover, DHM inhibited the protein expressions of β-catenin and GSK3β through the regulated extracellular-signal-regulated kinase (ERK)1/2 signaling pathway. By using the ERK1/2 activator, T-BHQ, reverted β-catenin and S100A4 protein expression and cell migration, which were reduced in response to DHM. In conclusion, our study indicated that DHM inhibited cell migration by reducing the S100A4 expression through the ERK1/2/β-catenin pathway in human cervical cancer cell lines.     

The PI3K/AKT Pathway Is Activated by HGF in NT2D1 Non-Seminoma Cells and Has a Role in the Modulation of Their Malignant Behavior

Overactivation of the c-MET/HGF system is a feature of many cancers. We previously reported that type II testicular germ cell tumor (TGCT) cells express the c-MET receptor, forming non-seminomatous lesions that are more positive compared with seminomatous ones. Notably, we also demonstrated that NT2D1 non-seminomatous cells (derived from an embryonal carcinoma lesion) increase their proliferation, migration, and invasion in response to HGF. Herein, we report that HGF immunoreactivity is more evident in the microenvironment of embryonal carcinoma biopsies with respect to seminomatous ones, indicating a tumor-dependent modulation of the testicular niche. PI3K/AKT is one of the signaling pathways triggered by HGF through the c-MET activation cascade. Herein, we demonstrated that phospho-AKT increases in NT2D1 cells after HGF stimulation. Moreover, we found that this pathway is involved in HGF-dependent NT2D1 cell proliferation, migration, and invasion, since the co-administration of the PI3K inhibitor LY294002 together with HGF abrogates these responses. Notably, the inhibition of endogenous PI3K affects collective cell migration but does not influence proliferation or chemotactic activity. Surprisingly, LY294002 administered without the co-administration of HGF increases cell invasion at levels comparable to the HGF-administered samples. This paradoxical result highlights the role of the testicular microenvironment in the modulation of cellular responses and stimulates the study of the testicular secretome in cancer lesions.