Cisplatin-resistant Cell Lines Research Articles

Long non-coding RNA NEAT1/miR-320b/MSI2 axis regulates cisplatin resistance in ovarian cancer

IntroductionOvarian cancer (OC) frequently occurs in postmenopausal women and it has higher mortality rate. Accumulating researches proved that long non-coding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) involved in the progression of chemoresistance in human OC. Here, the study aimed to investigate the partial molecular mechanism of OC chemoresistance.Material and methodsThe levels of NEAT1 and microRNA-320b (miR-320b) were measured by qRT-PCR. Western blot was carried out to determine the protein levels that used in this research. Cell viability was identified via Cell Counting Kit-8 (CCK-8). Transwell assay was employed to determine migration and invasion. The relationship between miR-320b and NEAT1 or MSI2 was clarified by dual-luciferase reporter assay, RNA immunoprecipitation (RIP) and RNA pull down assay. Also, a murine xenograft assay was used to explore the effect of NEAT1 on cisplatin resistance in OC in vivo.ResultsThe level of NEAT1 was significantly increased in cisplatin resistant OC cell lines. Downregulation of NEAT1 enhanced cisplatin sensibility in OVCAR-3/DDP and HEY/DDP cells. Furthermore, miR-320b was a target of NEAT1, and the effects of knockdown of NEAT1 on the cell viability, IC50 of cisplatin, migration and invasion in OVCAR-3/DDP and HEY/DDP were restored by the inhibitor of miR-320. In addition, miR-320b directly targeted MSI2 to regulate cisplatin sensibility in cisplatin resistant OC cells. In addition, downregulation of NEAT1 decreased cisplatin resistance in OC in vivo.ConclusionsNEAT1 regulated cisplatin resistance through NEAT1/miR-320b/MSI2 axis in OC, which might offer a novel therapy target for the chemotherapy of OC.

Amino Acid Conjugates of Aminothiazole and Aminopyridine as Potential Anticancer Agents: Synthesis, Molecular Docking and in vitro Evaluation.

PurposeThe development of resistance to available anticancer drugs is increasingly becoming a major challenge and new chemical entities could be unveiled to compensate this therapeutic failure. The current study demonstrated the synthesis of 2-aminothiazole [S3(a-d) and S5(a-d)] and 2-aminopyridine [S4(a-d) and S6(a-d)] derivatives that can target multiple cellular networks implicated in cancer development.MethodsBiological assays were performed to investigate the antioxidant and anticancer potential of synthesized compounds. Redox imbalance and oxidative stress are hallmarks of cancer, therefore, synthesized compounds were preliminarily screened for their antioxidant activity using DPPH assay, and further five derivatives S3b, S3c, S4c, S5b, and S6c, with significant antioxidant potential, were selected for investigation of in vitro anticancer potential. The cytotoxic activities were evaluated against the parent (A2780) and cisplatin-resistant (A2780CISR) ovarian cancer cell lines. Further, Molecular docking studies of active compounds were performed to determine binding affinities.ResultsResults revealed that S3c, S5b, and S6c displayed promising inhibition in cisplatin-resistant cell lines in comparison to parent cells in terms of both resistance factor (RF) and IC50 values. Moreover, S3c proved to be most active compound in both parent and resistant cell lines with IC50 values 15.57 µM and 11.52 µM respectively. Our docking studies demonstrated that compounds S3c, S5b, and S6c exhibited significant binding affinity with multiple protein targets of the signaling cascade.ConclusionAnticancer activities of compounds S3c, S5b, and S6c in cisplatin-resistant cell lines suggested that these ligands may contribute as lead compounds for the development of new anticancer drugs.

MicroRNA expression profiling and biomarker validation in treatment-naïve and drug resistant non-small cell lung cancer

BackgroundIn the absence of targetable mutations or immune checkpoints, cisplatin-doublet chemotherapy remains the standard of care in non-small cell lung cancer (NSCLC). Drug resistance has however become a significant clinical challenge. Exploring a role for small non-coding microRNAs (miRNA) as biomarker candidates in cisplatin resistant (CisR) lung cancer is lacking and warrants further investigation.MethodsmiRNA expression profiling was assessed in a panel of cisplatin sensitive and resistant NSCLC cell lines and validated by qPCR. Modulation of altered miRNAs was studied using antagomiRs and pre-miRs while functional assays were used to assess cisplatin response. The translational relevance of these miRNAs as potential biomarkers was assessed in serum and matched normal and tumour lung tissues from chemo-naïve NSCLC patients, in addition to xenograft formalin-fixed paraffin-embedded (FFPE) tumours derived from cisplatin sensitive and resistant cell lines.ResultsDifferential expression of a 5-miR signature (miR-30a-3p, miR-30b-5p, miR-30c-5p, miR-34a-5p, miR-4286) demonstrated their ability to distinguish between normal and tumour lung tissue and between NSCLC histologies. In squamous cell carcinoma (SqCC), tissue miRNA expression was associated with poor survival. miR-4286 showed promise as a blood-based diagnostic biomarker that could distinguish between adenocarcinoma and SqCC histologies. In a xenograft model of cisplatin resistance, using 7-9 week old female NOD/SCID mice (NOD.CB17-Prkdcscid/NCrCrl), a 5-miRNA panel showed altered expression between sensitive and resistant tumours.ConclusionsThis study identified a panel of miRNAs which may have diagnostic and prognostic potential as novel biomarkers in lung cancer and furthermore, may have a predictive role in monitoring the emergence of resistance to cisplatin.

PLGA nanoparticles delivering CPT-11 combined with focused ultrasound inhibit platinum resistant ovarian cancer.

BackgroundOvarian cancer cells show resistance to platinum drugs treatment, which brings a big challenge to clinical therapeutics. This study aimed to construct effective drug delivering nanoparticles specifically targeting ovarian cancer cell.MethodsPoly lactic-co-glycolic acid (PLGA) were used to form Nano-spheres by double emulsion method, and to deliver CPT-11. Connected with targeted LHRH-a molecules, their effects were tested by ovarian cancer cell A2780/DDP in vitro and in vivo.ResultsWe successfully constructed PLGA nanoparticles carrying LHRH-a (Luteinizing hormone releasing hormone analogue) and CPT-11 (irinotecan HCl trihydrate), which can specifically target LHRH receptor high expression ovarian cancer cell A2780/DDP (cisplatin). Combined with focused ultrasound in vitro, LHRH-a/CPT-11/PLGA nanoparticles significantly inhibited the proliferation of A2780/DDP cells (a cisplatin-resistant A2780 cell line), and the cells were obviously arrested at S phase. Both the mRNA expression and protein level of Caspase3 increased, while Bcl-2 and MMP2 declined, which promoted apoptosis. In vivo, LHRH-a/CPT-11/PLGA nanoparticles bind specifically with LHRH receptor on xenograft tumors of A2780/DDP. With focused ultrasound, LHRH-a/CPT-11/PLGA nanoparticles inhibited the growth of A2780/DDP xenograft tumors significantly. The expression level of VEGF, Bcl-2 and MMP2 reduced, while Caspase3 increased in tumors.ConclusionsCPT-11 delivering PLGA nanoparticles with LHRH-a specifically target ovarian cancer cell A2780/DDP, and work locally when combined with focused ultrasound. They increase local drug concentration and reduce side effects. This research may provide a new effective therapeutic strategy for recurrent platinum resistant ovarian cancer.

The m6A methyltransferase METTL3 regulates autophagy and sensitivity to cisplatin by targeting ATG5 in seminoma.

BackgroundOur previous work shows Autophagy enhanced resistance to cisplatin in seminoma. The expression of the N6-methyladenosine (m6A) methyltransferases METTL3 was significantly increased in the cisplatin-resistant TCam-2 cell line of seminoma. We aimed to investigate the role of m6A methylation in autophagy and the chemosensitivity of seminoma cells.MethodsPlasmid and siRNA were used to overexpress and knockdown METTL3. Autophagy was detected by western blot and immunofluorescence, respectively. The expression of downstream targets of METTL3 was detected by quantitative real-time PCR (qRT-PCR) and western blot, and the m6A level of them was detected by MeRIP-qPCR. Chemosensitivity of the TCam-2 cell line was identified through MTT assay.ResultsUpon METTL3 overexpression, autophagy of TCam-2 cell line was enhanced and its sensitivity to cisplatin was decreased. The use of autophagy inhibitors 3-methyladenine (3-MA) could reverse the protective effect of METTL3 on TCam-2 cells. We found that the up-regulation of METTL3 could increase the m6A modification level of ATG5 transcript, thus increased expression of ATG5. Moreover, knockdown of ATG5 reduced METTL3-induced autophagy, suggesting that ATG5 was a potential target for METTL3 to promote autophagy.ConclusionsIn summary, our research unveiled the unique mechanism by which m6A methylation regulates autophagy and chemosensitivity of the TCam-2 cell line and METTL3 was a potential target to overcome the cisplatin resistance of seminoma.

LINC00184 Promotes Ovarian Cancer Cells Proliferation and Cisplatin Resistance by Elevating CNTN1 Expression via Sponging miR-1305.

ObjectiveCisplatin resistance is one of the main reasons for treatment failure in ovarian cancer (OC). Here, the effects of LINC00184 on cisplatin-resistant OC were studied.Patients and MethodsLINC00184, miR-1305 and CNTN1 expression in tissues from 70 OC patients was determined by qRT-PCR, in situ hybridization and Western blot. OC cell lines and OC cisplatin-resistant cell lines were cultured. Cells were transfected using Lipofectamine 2000 and treated with 100 nM cisplatin. Cell proliferation and apoptosis were researched by the CCK-8 assay and flow cytometry. A dual-luciferase reporter gene assay and RNA pull-down were performed to explore the relationship between two genes. LINC00184, miR-1305 and CNTN1 expression in cells was detected by qRT-PCR and Western blot. An in vivo experiment was conducted using nude mice. Ki67 and CNTN1 expression and apoptosis of xenograft tumors were investigated using immunohistochemistry and a TUNEL assay.ResultsLINC00184 was up-regulated in OC clinical tissues and OC cells, especially in cisplatin-resistant OC patients and cells (p<0.01 or p<0.0001). LINC00184 overexpression significantly enhanced OC cell proliferation and cisplatin resistance, and inhibited OC cell apoptosis (p<0.05 or p<0.01). LINC00184 elevated CNTN1 expression via sponging miR-1305. LINC00184 overexpression markedly exacerbated the malignant phenotype of OC cells and cisplatin-resistant OC cells via the miR-1305/CNTN1 axis (p<0.01). Silencing of LINC00184 significantly suppressed OC cell growth and cisplatin resistance in vivo (p<0.01). LINC00184 silencing inhibited Ki67 and CNTN1 expression and promoted apoptosis of xenograft tumors. CNTN1 overexpression promoted proliferation and cisplatin resistance, and reduced apoptosis of OC cells (p<0.05 or p<0.01).ConclusionLINC00184 promoted OC cell proliferation and cisplatin resistance by elevating CNTN1 expression via sponging miR-1305.

Arene Ruthenium(II) Complexes Bearing the κ-P or κ-P,κ-S Ph2P(CH2)3SPh Ligand

Neutral [Ru(η6-arene)Cl2{Ph2P(CH2)3SPh-κP}] (arene = benzene, indane, 1,2,3,4-tetrahydronaphthalene: 2a, 2c and 2d) and cationic [Ru(η6-arene)Cl(Ph2P(CH2)3SPh-κP,κS)]X complexes (arene = mesitylene, 1,4-dihydronaphthalene; X = Cl: 3b, 3e; arene = benzene, mesitylene, indane, 1,2,3,4-tetrahydronaphthalene, and 1,4-dihydronaphthalene; X = PF6: 4a–4e) complexes were prepared and characterized by elemental analysis, IR, 1H, 13C and 31P NMR spectroscopy and also by single-crystal X-ray diffraction analyses. The stability of the complexes has been investigated in DMSO. Complexes have been assessed for their cytotoxic activity against 518A2, 8505C, A253, MCF-7 and SW480 cell lines. Generally, complexes exhibited activity in the lower micromolar range; moreover, they are found to be more active than cisplatin. For the most active ruthenium(II) complex, 4b, bearing mesitylene as ligand, the mechanism of action against 8505C cisplatin resistant cell line was determined. Complex 4b induced apoptosis accompanied by caspase activation.

MicroRNA-125a-5p targets LIM kinase 1 to inhibit cisplatin resistance of cervical cancer cells.

Cervical cancer (CC), also known as invasive cervical carcinoma, is one of the most common gynecologic malignancies. The aim of the present study was to investigate the function of microRNA (miR)-125a-5p on CC progression and cisplatin (DDP) resistance. For this purpose, reverse transcription-quantitative PCR (RT-qPCR) was used to assess the expression of miR-125a-5p and LIMK1 in CC tissues, corresponding normal tissues and cells (human CC cell lines: C-33A, CaSKi; human cervical epithelial cells: HUCEC). Cisplatin (DDP) resistant cervical cancer cell lines were established (C-33A/DDP and CaSKi/DDP cell lines). RT-qPCR results demonstrated that miR-125a-5p or LIM kinase 1 (LIMK1) expression was downregulated or upregulated in C-33A/DDP and CaSKi/DDP cells, respectively. MTT assay, flow cytometry analysis and Western blotting were employed to detect the proliferation, apoptosis rate, IC50 of DDP and the expression of drug resistance-related proteins (P-glycoprotein and glutathione S-transferase-π). The targeting relationship between miR-125a-5p and LIMK1 was confirmed by the TargetScan database and dual-luciferase reporter gene assay. In CC tissues and cell lines, compared with normal tissues or HUCEC, miR-125a-5p expression was downregulated and LIMK1 expression was upregulated. The transfection with miR-125a-5p mimics decreased the proliferation of CaSKi/DDP cells, increased the apoptosis rate, reduced the IC50 of DDP, and downregulated the expression of drug resistance-related proteins; conversely, LIMK1 overexpression decreased the apoptosis rate, increased the IC50 of DDP, and upregulated the expression of drug resistance-related proteins. The luciferase reporter gene assay demonstrated that miR-125a-5p targeted and negatively regulated LIMK1. miR-125a-5p could partially reverse the effect of LIMK1 on the proliferation, apoptosis, IC50 of DDP and the expressions of drug resistance-related proteins. The findings of the present study indicated that miR-125a-5p sensitizes CC cells to DDP by targeting LIMK1, hence increasing the anticancer efficacy of cisplatin.

Design, Synthesis and Pharmacological Evaluation of Three Novel Dehydroabietyl Piperazine Dithiocarbamate Ruthenium (II) Polypyridyl Complexes as Potential Antitumor Agents: DNA Damage, Cell Cycle Arrest and Apoptosis Induction.

The use of cisplatin is severely limited by its toxic side-effects, which has spurred chemists to employ different strategies in the development of new metal-based anticancer agents. Here, three novel dehydroabietyl piperazine dithiocarbamate ruthenium (II) polypyridyl complexes (6a–6c) were synthesized as antitumor agents. Compounds 6a and 6c exhibited better in vitro antiproliferative activity against seven tumor cell lines than cisplatin, they displayed no evident resistance in the cisplatin-resistant cell line A549/DPP. Importantly, 6a effectively inhibited tumor growth in the T-24 xenograft mouse model in comparison with cisplatin. Gel electrophoresis assay indicated that DNA was the potential targets of 6a and 6c, and the upregulation of p-H2AX confirmed this result. Cell cycle arrest studies demonstrated that 6a and 6c arrested the cell cycle at G1 phase, accompanied by the upregulation of the expression levels of the antioncogene p27 and the down-regulation of the expression levels of cyclin E. In addition, 6a and 6c caused the apoptosis of tumor cells along with the upregulation of the expression of Bax, caspase-9, cytochrome c, intracellular Ca2+ release, reactive oxygen species (ROS) generation and the downregulation of Bcl-2. These mechanistic study results suggested that 6a and 6c exerted their antitumor activity by inducing DNA damage, and consequently causing G1 stage arrest and the induction of apoptosis.

Targeted biologic inhibition of both tumor cell-intrinsic and intercellular CLPTM1L/CRR9-mediated chemotherapeutic drug resistance

Recurrence of therapy-resistant tumors is a principal problem in solid tumor oncology, particularly in ovarian cancer. Despite common complete responses to first line, platinum-based therapies, most women with ovarian cancer recur, and eventually, nearly all with recurrent disease develop platinum resistance. Likewise, both intrinsic and acquired resistance contribute to the dismal prognosis of pancreatic cancer. Our previous work and that of others has established CLPTM1L (cleft lip and palate transmembrane protein 1-like)/CRR9 (cisplatin resistance related protein 9) as a cytoprotective oncofetal protein that is present on the tumor cell surface. We show that CLPTM1L is broadly overexpressed and accumulated on the plasma membrane of ovarian tumor cells, while weakly or not expressed in normal tissues. High expression of CLPTM1L is associated with poor outcome in ovarian serous adenocarcinoma. Robust re-sensitization of resistant ovarian cancer cells to platinum-based therapy was achieved using human monoclonal biologics inhibiting CLPTM1L in both orthotopic isografts and patient-derived cisplatin resistant xenograft models. Furthermore, we demonstrate that in addition to cell-autonomous cytoprotection by CLPTM1L, extracellular CLPTM1L confers resistance to chemotherapeutic killing in an ectodomain-dependent fashion, and that this intercellular resistance mechanism is inhibited by anti-CLPTM1L biologics. Specifically, exosomal CLPTM1L from cisplatin-resistant ovarian carcinoma cell lines conferred resistance to cisplatin in drug-sensitive parental cell lines. CLPTM1L is present in extracellular vesicle fractions of tumor culture supernatants and in patients’ serum with increasing abundance upon chemotherapy treatment. These findings have encouraging implications for the use of anti-CLPTM1L targeted biologics in the treatment of therapy-resistant tumors.

LncRNA LHFPL3-AS1 Promotes Oral Squamous Cell Carcinoma Growth and Cisplatin Resistance Through Targeting miR-362-5p/CHSY1 Pathway

BackgroundOral squamous cell carcinoma (OSCC) is a common oral cancer. The current study aims to elucidate the potential roles of long noncoding RNA (lncRNA) LHFPL3-AS1 in OSCC development.MethodsGene expression was measured by qRT-PCR in tumor tissues and cell lines. Loss-of-function assays were performed to analyze the effects of LHFPL3-AS1 on malignant behaviors. Bioinformatics analysis was conducted to explore the downstream signaling pathway of LHFPL3-AS1 in OSCC.ResultsLHFPL3-AS1 was highly expressed in OSCC tissues and cell lines. LHFPL3-AS1 was upregulated in cisplatin-resistant tumor cell lines. LHFPL3-AS1 level was correlated with survival rate. LHFPL3-AS1 knockdown suppressed OSCC proliferation, migration and invasion. LHFPL3-AS1 downregulation reduced cisplatin resistance of OSCC cells. LHFPL3-AS1 was the competing endogenous RNA (ceRNA) for miR-194-5p to enhance CHSY1 expression.ConclusionLHFPL3-AS1/miR-362-5p/CHSY1 signaling pathway plays essential roles in regulating OSCC development and cisplatin resistance.

Ru complexes containing Cp, mPTA and natural purine bases (mPTA = methyl-N-1,3,5-triaza-7-phosphaadamantane): Evaluation of their antiproliferative activity, solubility and redox properties

Complexes [RuCp(Adeninate–κN9)(mPTA)2](Cl0.5)(CF3SO3)2.5·H2O (1·H2O), [RuCp(Guaninate-κN7)(mPTA)2](CF3SO3)2·H2O (2·H2O), [RuCp(Theophyllinate-κN7)(mPTA)2](CF3SO3)2·1.5H2O (3·1.5H2O) and [RuCp(Pur-κN)(mPTA)(PPh3)](CF3SO3) (4–6) (Pur = Adeninate, Guaninate, Teophyllinate; mPTA = N-methyl −1,3,5-triaza-7-phosphaadamantane) have been synthesized and characterized. Structure of complexes 1·H2O and 3·1.5H2O were determined by single-crystal X-ray diffraction. Solubility in water, Log P, electrochemical properties and antiproliferative activities of the complexes (against cisplatin-sensitive T2 and cisplatin-resistant SKOV3 cell lines) have been assessed and discussed.

Testicular Germ Cell Tumors Acquire Cisplatin Resistance by Rebalancing the Usage of DNA Repair Pathways.

Simple SummaryGerm cell tumors are a model of curable solid tumors due to their unique sensitivity to cisplatin-based chemotherapy. Patients are typically young adults, and despite high cure rate, about 20% of them do not achieve remission or relapse, and 50% of them succumb to the disease. The mechanisms behind their resistance to therapy are largely unknown. By using Testicular Germ Cell Tumor (TGCT) cell lines as a model, we investigated the mechanism of acquired resistance to cisplatin. We demonstrated that resistance occurred by a fine modulation of the DNA repair pathway choice. Namely, in resistant cells, repair of double-strand breaks by non-homologous end joining was dampened by the reduced expression of TP53-binding protein 1 (53BP1) and DNA-dependent protein kinase (DNA-PKcs). However, cisplatin-induced damage was repaired efficiently by homologous recombination. Additionally, we demonstrate that pharmacological inhibition of poly (ADP-ribose) polymerase (PARP) combined with cisplatin had an additive/synergistic effect on cisplatin-resistant cells, which represents the proof of concept for introducing PARP inhibitors in salvage therapy.Despite germ cell tumors (GCTs) responding to cisplatin-based chemotherapy at a high rate, a subset of patients does not respond to treatment and have significantly worse prognosis. The biological mechanisms underlying the resistance remain unknown. In this study, by using two TGCT cell lines that have acquired cisplatin resistance after chronic exposure to the drug, we identified some key proteins and mechanisms of acquired resistance. We show that cisplatin-resistant cell lines had a non-homologous end-joining (NHEJ)-less phenotype. This correlated with a reduced basal expression of TP53-binding protein 1 (53BP1) and DNA-dependent protein kinase (DNA-PKcs) proteins and reduced formation of 53BP1 foci after cisplatin treatment. Consistent with these observations, modulation of 53BP1 protein expression altered the cell line’s resistance to cisplatin, and inhibition of DNA-PKcs activity antagonized cisplatin cytotoxicity. Dampening of NHEJ was accompanied by a functional increase in the repair of DNA double-strand breaks (DSBs) by the homologous recombination repair pathway. As a result, cisplatin-resistant cells were more resistant to PARP inhibitor (PARPi) monotherapy. Moreover, when PARPi was given in combination with cisplatin, it exerted an additive/synergistic effect, and reduced the cisplatin dose for cytotoxicity. These results suggest that treatment of cisplatin-refractory patients may benefit from low-dose cisplatin therapy combined with PARPi.

Cisplatin-Resistant Gastric Cancer Cells Promote the Chemoresistance of Cisplatin-Sensitive Cells via the Exosomal RPS3-Mediated PI3K-Akt-Cofilin-1 Signaling Axis.

Cisplatin is an important agent in first-line chemotherapy against gastric cancer (GC). However, consequential drug resistance limits its effectiveness for the treatment of GC. In this study, a cisplatin resistant gastric cancer cell line SGC7901R was determined by LC-MS/MS with increased exosomal levels of RPS3 protein. SGC7901R cell-derived exosomes were readily taken up by cisplatin-sensitive SGC7901S cells, thus triggering off a phenotype of chemoresistance in the receptor cells. Subsequently, it was demonstrated that exosomal RPS3 was essential for inducing chemoresistance of receptor cells as shown by the acquisition of this phenotype in SGC7901S cells with enforced expression of RPS3. Further mechanism study demonstrated that cisplatin-resistant gastric cancer cell-derived exosomal RPS3 enhanced the chemoresistance of cisplatin-sensitive gastric cancer cells through the PI3K-Akt-cofilin-1 signaling pathway. All these findings demonstrated that cisplatin-resistant gastric cancer cells communicate with sensitive cells through the intercellular delivery of exosomal RPS3 and activation of the PI3K-Akt-cofilin-1 signaling pathway. Targeting exosomal RPS3 protein in cisplatin-resistant gastric cancer cells may thus be a promising strategy to overcome cisplatin resistance in gastric cancer.

Effects of FER1L4-miR-106a-5p/miR-372-5p-E2F1 regulatory axis on drug resistance in liver cancer chemotherapy

Liver cancer presents a challenge in today’s healthcare system. This study aimed at investigating the effects of Fer-1 like family member 4 (FER1L4) on chemotherapy resistance and liver cancer development by using clinically collected liver cancer tissues and commercially purchased human liver cancer cisplatin-resistant cell line HUH-7/DDP. Bioinformatics analysis, dual luciferase reporter gene assay, and RNA pull-down were applied to predict and verify the possible binding relationships. The expressions of FER1L4, E2F transcription factor 1 (E2F1), microRNA-106a-5p (miR-106a-5p), or miR-372-5p were altered in the cells, followed by flow cytometry, Cell Counting Kit-8 (CCK-8), and Transwell assays to evaluate apoptotic, proliferative, and invasive abilities in vitro and nude mice xenografts to observe tumor growth in vivo. FER1L4 was highly expressed and miR-106-5p and miR-372-5p were poorly expressed in tumor cells and tissues. FER1L4 knockdown or the overexpression of miR-106-5p and miR-372-5p inhibited the cancerous cell proliferation and invasion while promoting apoptosis. FERIL4 silencing increased the miR-106-5p/miR-372-5p expression to inhibit the E2F1-activated nuclear factor κB (NF-κB) pathway. Besides, overexpressing FER1L4 led to an increased tumor growth in nude mice, which was reversed by the NF-κB inhibitor pyrollidine dithiocarbamate (PDTC). In conclusion, the results indicated that FER1L4 could inhibit the expression of miR-106a-5p/miR-372-5p, to activate E2F1-mediated NF-κB pathway, leading to drug resistance in liver cancer.

Distinct Side Population Cell Subtypes Have Different Stemness Levels in Human Ovarian Cancer Cells.

The stemness of different side population (SP) cell subtypes in ovarian cancer cells was studied, and the heterogeneity of ovarian cancer stem cells was analyzed. The cisplatin-resistant human serous ovarian cancer cell line C13 was stained with the bisbenzimide Hoechst 33342. A flow cytometry-based fluorescence-activated sorting method was used to obtain lower-SP (LSP) cells, upper-SP (USP) cells, and non-SP cells (NSP) based on their sensitivity to the staining time and Hoechst dye concentration. The sphere-forming capability, expression levels of stem cell markers, resistance to high concentrations of cisplatin, and subcutaneous tumorigenicity in NOD/SCID mice of the different cell subtypes were evaluated. The C13 cells contained SP cells with stemness characteristics, and the LSP cell subtype expressed higher levels of stem cell markers, had higher in vitro sphere-forming capability, higher cisplatin resistance and higher in vivo subcutaneous tumorigenesis than USP cells (P<0.05). NSP cells had no stemness. In conclusion, different subtypes of ovarian cancer SP cells have different stemness levels, and ovarian cancer stem cells may be heterogeneous.

Targeting YTHDF1 effectively re-sensitizes cisplatin-resistant colon cancer cells by modulating GLS-mediated glutamine metabolism

Colorectal cancer (CRC) has a high mortality rate and poor prognosis. Despite chemotherapeutic agents such as cisplatin, which has achieved a better prognosis and survival rate against cancer, drug resistance leads to significant challenges. Accumulating evidence suggests that YTHDF1, the N6-methyladenosine (m6A) “reader,” is an important regulator in tumor progresses. Herein, we report that YTHDF1 was significantly upregulated in human colon tumors and cell lines. Overexpression of YTHDF1 decreased the cisplatin sensitivity of colon cancer cells. From the established cisplatin-resistant CRC cell line (LoVo CDDP R), we detected that YTHDF1 was significantly upregulated in cisplatin-resistant CRC cells. Intriguingly, RNA sequencing (RNA-seq) results revealed that glutamine metabolism enzymes were clearly upregulated in LoVo CDDP R cells. Glutamine uptake, that is, glutaminase (GLS) activity, was upregulated in LoVo CDDP R cells. Furthermore, bioinformatics analysis indicated that the 3′ UTR of GLS1 contained a putative binding motif of YTHDF1, and an interaction was further validated by a protein-RNA interaction assay (RNA immunoprecipitation [RIP]). Furthermore, we demonstrated that YTHDF1 promoted protein synthesis of GLS1. Inhibiting GLS1 effectively synergizes with cisplatin to induce colon cancer cell death. Finally, that YTHDF1 mediated cisplatin through the GLS1-glutamine metabolism axis was validated by an in vivo xenograft mouse model. In summary, our study reveals a new mechanism for YTHDF1-promoted cisplatin resistance, contributing to overcoming chemoresistant colon cancers.

EHHADH contributes to cisplatin resistance through regulation by tumor-suppressive microRNAs in bladder cancer

BackgroundCisplatin-based chemotherapy is recommended as the primary treatment for advanced bladder cancer (BC) with unresectable or metastatic disease. However, the benefits are limited due to the acquisition of drug resistance. The mechanisms of resistance remain unclear. Although there are some reports that some molecules are associated with cisplatin resistance in advanced BC, those reports have not been fully investigated. Therefore, we undertook a new search for cisplatin resistance-related genes targeted by tumor suppressive microRNAs as well as genes that were downregulated in cisplatin-resistant BC cells and clinical BC tissues.MethodsFirst, we established cisplatin-resistant BOY and T24 BC cell lines (CDDP-R-BOY, CDDP-R-T24). Then, Next Generation Sequence analysis was performed with parental and cisplatin-resistant cell lines to search for the microRNAs responsible for cisplatin resistance. We conducted gain-of-function analysis of microRNAs and their effects on cisplatin resistance, and we searched target genes comprehensively using Next Generation mRNA sequences.ResultsA total of 28 microRNAs were significantly downregulated in both CDDP-R-BOY and CDDP-R-T24. Among them, miR-486-5p, a tumor suppressor miRNA, was negatively correlated with the TNM classification of clinical BC samples in The Cancer Genome Atlas (TCGA) database. Transfection of miRNA-486-5p significantly inhibited cancer cell proliferation, migration, and invasion, and also improved the cells’ resistance to cisplatin. Among the genes targeted by miRNA-486-5p, we focused on enoyl-CoA, hydratase/3-hydroxyacyl CoA dehydrogenase (EHHADH), which is involved in the degradation of fatty acids. EHHADH was directly regulated by miRNA-486-5p as determined by a dual-luciferase reporter assay. Loss-of-function study using EHHADH si-RNA showed significant inhibitions of cell proliferation, migration, invasion and the recovery of cisplatin sensitivity.ConclusionIdentification of EHHADH as a target of miRNA-486-5p provides novel insights into the potential mechanisms of cisplatin resistance in BC.

Targeting Germ Cell Tumors with the Newly Synthesized Flavanone-Derived Compound MLo1302 Efficiently Reduces Tumor Cell Viability and Induces Apoptosis and Cell Cycle Arrest

Less toxic treatment strategies for testicular germ cell tumor (TGCT) patients are needed, as overtreatment is a concern due to the long-term side effects of platin-based chemotherapy. Although clinical benefit from classical hypomethylating agents has to date been limited, TGCTs show an abnormal DNA methylome indicating the potential of treating TGCTs with hypomethylating drugs. We tested, for the first time in TGCT cell lines, a new synthetic flavonoid compound (MLo1302) from the 3-nitroflavanone family of DNA methyltransferase (DNMT) inhibitors. We show that MLo1302 reduces cell viability (including of cisplatin resistant cell line NCCIT-R), with IC50s (inhibitory concentration 50) within the nanomolar range for NCCIT and NTERA-2 cells, and proved its cytotoxic effect. Exposure to MLo1302 reduced DNMT protein expression, similar to decitabine, and showed a partial effect in cell differentiation, reducing protein expression of pluripotency markers. RT2 profiler expression array indicated several dysregulated targets, related to activation of apoptosis, differentiation, and cell cycle arrest. We validated these data by showing increased apoptosis, increased protein expression of cleaved caspase 8 and activated caspase 2, and reduced proliferation (BrdU assay), with increase in CDKN1A and decrease in MIB-1 expression. Therefore, synthetic drugs designed to target DNA methylation in cells may uncover effective treatments for TGCT patients.

Knockdown of USP9X reverses cisplatin resistance by decreasing β-catenin expression in nasopharyngeal carcinoma cells.

In various cancers, abnormal USP9X expression is involved in tumorigenesis, progression, apoptosis, and metastasis. However, the relationship between USP9X abnormal expression and cisplatin resistance in nasopharyngeal carcinoma (NPC) cells remains unclear. Using qRT-PCR and western blot, we detected the expressions of USP9X and β-catenin in NPC cells. The effects of USP9X on cisplatin resistance, proliferation, apoptosis, and metastasis were examined by CCK-8 assay, flow cytometry, wound-healing assay, and Transwell chamber assay. Co-IP assay, qRT-PCR, and western blot were performed to explore the detailed molecular mechanism of USP9X-β-catenin and its effect on the protein levels of MDR1, MRP2, Bcl-2, Bax, MMP2, and MMP9. We found that USP9X and β-catenin expressions in cisplatin-resistant cell lines (HNE1/DDP) were much higher than cisplatin-sensitive cell lines (HNE1) at both mRNA and protein levels. Co-IP assay demonstrated that USP9X was immunoprecipitated with β-catenin in NPC cells. Knockdown of USP9X was able to partially reverse cisplatin resistance, increased cisplatin-induced apoptosis, and decreased the capacities of proliferation, migration, and invasion. Overexpression of USP9X can increase cisplatin resistance in NPC cells. Moreover, knockdown of USP9X expression can significantly reduce the expressions of MDR1, MRP2, Bcl-2, MMP2, and MMP9, but significantly increased the expression of Bax. These findings indicate that USP9X high expression plays a significant part in cisplatin resistance of NPC. This study elucidated the possible mechanism of cisplatin resistance in NPC cells and may have implications for the therapeutic reversal of cisplatin resistance.