Vincristine Resistance Research Articles

Beneficial Effect of Microrna-155 in Vincristine Response and Clinical Outcome in Especially GCB Diffuse Large B-Cell Lymphoma

▪R-CHOP is standard treatment for previously untreated diffuse large B-cell lymphoma (DLBCL). Approximately 35-40% of patients relapse following R-CHOP due to drug-specific molecular resistance, and outcome with salvage therapy remain poor. Several clinical trials have tried to improve the R-CHOP regimen by dose adjustments and add on drugs, but with limited clinical benefit, emphasizing that improved understanding of R-CHOP resistance are still relevant and highly needed. Vincristine is essential to the efficacy of R-CHOP, and despite its use for decades, little is known about determinants of resistance in the treatment of DLBCL, which is important when attempting to improve outcome.We hypothesized that miRNAs are involved in the underlying molecular mechanisms by which Vincristine resistance work and that miRNAs hold potential as biomarkers of Vincristine response. By analyses of miRNA microarrays from DLBCL cell lines with different Vincristine sensitivity, we identified lower miR-155 expression in resistant cell lines.The hypothesis was further studied by two different approaches; first, by experimental manipulations of miR-155 expression by lentiviral vectors in DLBCL cell lines, studying the direct impact of miR-155 in Vincristine response, and secondly, by clinical outcome analysis of the prognostic impact of miR-155 expression in two independent DLBCL cohorts.Our results document that miR-155 is directly involved in Vincristine response in DLBCL cells since over-expression of miR-155 increased the Vincristine sensitivity, especially in GCB classified cell lines. Consistently, when miR-155 was downregulated or knocked-out by the CRISPR-Cas9 technology the cells became more resistant.Prognostic impact of miR-155 was evaluated in an in-house cohort of 69 R-CHOP treated patients having miR-155 expression determined by RT-qPCR and in an independent DLBCL meta cohort of 586 patients, who had the miR-155 precursor MIR155HG measured by microarray. Survival analysis was performed overall and according to ABC/GCB subclasses. miR-155 expression did not correlate with clinical outcome after R-CHOP treatment in the entire DLBCL cohort or restricted to ABC classified patients. However, we observed that GCB classified patients with low miR-155 levels had significantly shorter overall and progression-free survival than GCB patients with high miR-155 expression levels (Figure 1). Multivariate Cox regression showed that miR-155 expression added significant and independent prognostic information to the already well-established international prognostic index (IPI) when the analysis was restricted to the GCB subclass.In summary, experimentally we confirmed a direct link between miR-155 and Vincristine response in DLBCL overall and in particular GCB DLBCL. This association is supported by prognostic evaluation in two clinical cohorts treated with R-CHOP, that documents an inferior clinical outcome in GCB classified patients with low miR-155 expression. This suggests that the role of miR-155 in Vincristine response is important enough to affect overall- and progression-free survival especially within the GCB subclass. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

TUFT1 Facilitates Metastasis, Stemness, and Vincristine Resistance in Colorectal Cancer via Activation of PI3K/AKT Pathway.

Since the incidence and mortality of colorectal cancer (CRC) are increasing in recent years, the research on the pathogenesis of colorectal cancer has attracted more and more attention. Here, our results confirmed that the mRNA expression level and proteins accumulation of TUFT1 were significantly increased in CRC tissues from late-stage CRC patients (III + IV) (p < 0.001), indicated by qPCR and IHC assay. The TUFT1 expression was positively correlated with tumor stage by analyzing 126 specimens from CRC patients. Next, we found that up-regulation of TUFT1 enhanced the migration and invasion of LoVo cells, whereas the down-regulation of TUFT1 observably weakened the migration and invasion of SW837 cells, indicating that TUFT1 promotes the metastasis of CRC cells. In addition, TUFT1 overexpression increased the number of mammary spheres and vincristine resistance of LoVo cells by sphere formation assay and measuring the IC50 value, suggesting the TUFT1 promotes stemness and the vincristine resistance of CRC cells. Finally, we found that TUFT1 overexpression increased p-AKT in LoVo cells, while down-regulation of TUFT1 decreased the p-AKT levels in SW837 cells. Therefore, we determined that the function of TUFT1 in CRC depends on PI3K/AKT pathway. Taken together, these data demonstrated that TUFI1 facilitates metastasis, stemness, and vincristine resistance of colorectal cancer cells via activation of PI3K/AKT pathway, which might act as a promising therapeutic target for CRC.

Retraction: Long non-coding RNA GACAT1 alleviates doxorubicin and vincristine resistance through a PTEN/AKT/mTOR/S6K1 regulatory pathway in gastric cancer.

Retraction of ‘Long non-coding RNA GACAT1 alleviates doxorubicin and vincristine resistance through a PTEN/AKT/mTOR/S6K1 regulatory pathway in gastric cancer’ by Hengxuan Ding et al., RSC Adv., 2019, 9, 8048–8055, DOI: 10.1039/C8RA10030F.

Autocrine IL-6/STAT3 signaling aids development of acquired drug resistance in Group 3 medulloblastoma

Medulloblastoma (MB) is a high-grade pediatric brain malignancy that originates from neuronal precursors located in the posterior cranial fossa. In this study, we evaluated the role of STAT3 and IL-6 in a tumor microenvironment mediated drug resistance in human MBs. We established that the Group 3 MB cell line, Med8A, is chemosensitive (hence Med8A-S), and this is correlated with a basal low phosphorylated state of STAT3, while treatment with IL-6 induced robust increases in pY705-STAT3. Via incremental selection with vincristine, we derived the stably chemoresistant variant, Med8A-R, that exhibited multi-drug resistance, enhanced IL-6 induced pY705-STAT3 levels, and increased IL6R expression. Consequently, abrogation of STAT3 or IL6R expression in Med8A-R led to restored chemosensitivity to vincristine, highlighting a prominent role for canonical IL-6/STAT3 signaling in acquired drug resistance. Furthermore, Med8A-S subjected to conditioning exposure with IL-6, termed Med8A-IL6+ cells, exhibited enhanced vincristine resistance, increased expression of pY705-STAT3 and IL6R, and increased secretion of IL-6. When cocultured with Med8A-IL6+ cells, Med8A-S cells exhibited increased pY705-STAT3 and increased IL-6 secretion, suggesting a cytokine feedback loop responsible for amplifying STAT3 activity. Similar IL-6 induced phenomena were also observed in the Group 3 MB cell lines, D283 and D341, including increased pY705-STAT3, drug resistance, IL-6 secretion and IL6R expression. Our study unveiled autocrine IL-6 as a promoter of STAT3 signaling in development of drug resistance, and suggests therapeutic benefits for targeting the IL-6/STAT3 signaling axis in Group 3 MBs.

Long noncoding RNA H19 promotes vincristine resistance in multiple myeloma by targeting Akt

Multiple myeloma is a malignant proliferation of plasma cells that results from a single clone. Manifestations include bone pain or fractures, kidney failure, susceptibility to infection, anemia, and hypercalcemia. To investigate the relationship between vincristine (VCR) resistance and Long noncoding RNA H19 (Lnc-RNA H19) in multiple myeloma (MM) this experiment was set up. For this aim, RT-PCR was used to detect the expression of lnc-RNA H19 in 60 MM patients from No.215 Hospital of Shaanxi Nuclear Industry and 50 healthy controls, and further detected the expression of related genes in myeloma cell lines and VCR myeloma resistant strains. MTT assay, flow cytometry assay, western-blotting assay and luciferase assay were used to analyze the growth, apoptosis and protein phosphorylation levels of drug-resistant RPMI 8226/VCR and RPMI 8226 cells after VCR treatment and plasmid transprinting. Results showed that the relative expression of lnc-RNA H19 was significantly increased in MM patients and drug-resistant strains RPMI 8226-VCR (****p&lt;0.0001), while apoptosis of various MM cell lines increased after VCR treatment, while apoptosis of RPMI 8226-VCR was significantly decreased (***p&lt;0.001). Lnc-RNA H19 overexpression plasmid pcdna3.1-h19 and Akt overexpression plasmid pcdna3.1-akt decreased apoptosis in RPMI 8226 cell lines without VCR resistance (***p&lt;0.001), while the recombination of siRNA-h19 and siRNA-akt plasmid increased apoptosis in RPMI 8226-VCR (***p&lt;0.001). The lnc-RNA H19/Akt pathway is closely related to the occurrence of VCR resistance in MM cells, and the down-regulation of H19 can significantly improve the sensitivity of VCR in MM.

MicroRNA-222-Mediated VHL Downregulation Facilitates Retinoblastoma Chemoresistance by Increasing HIF1α Expression.

PurposeRetinoblastoma (RB) is the most common primary intraocular tumor in children. Chemoresistance is the major obstacle for treatment of these tumors. This study aims to determine whether or not downregulating microRNA-222 (miR-222) could serve as a potential therapeutic target for preventing chemoresistance in RB treatment.MethodsDifferentially expressed miR-222 in RB samples and its downstream target genes were predicted using bioinformatics methods. The expression of miR-222 was altered by mimic or inhibitor to examine its role in RB cell in response to the chemotherapeutic agent vincristine (VCR). Further bioinformatic analysis predicted involvement of the stability of hypoxia-inducible factor 1α (HIF1α) protein in regulation of the von Hippel–Lindau (VHL) tumor suppressor, followed by characterization of the effect of VHL on the ubiquitin–proteasome degradation of HIF1α. Next, VHL or HIF1α was overexpressed to determine their effects on RB cell activities after VCR treatment. In vivo assays were performed on nude mice to further verify the in vitro results.ResultsmiR-222 is highly expressed in RB tissues and cells and was found to facilitate resistance of RB cells to VCR. Of note, miR-222 specifically bound to and negatively regulated VHL. VHL could inhibit the stability of HIF1α and promote the degradation of ubiquitin–proteasome, thus reducing HIF1α expression to attenuate VCR resistance in RB cells. Moreover, inhibition of miR-222 in combination with VCR suppressed tumor formation in nude mice.ConclusionsmiR-222 promotes the expression of HIF1α by targeting VHL, thus accelerating the resistance of RB cells to the chemotherapeutic agent VCR.

Overcoming vincristine resistance in cancer: Computational design and discovery of piperine-inspired P-glycoprotein inhibitors.

P-glycoprotein (P-gp)/MDR-1 plays a major role in the development of multidrug resistance (MDR) by pumping the chemotherapeutic drugs out of the cancer cells and reducing their efficacy. A number of P-gp inhibitors were reported to reverse the MDR when co-administered with chemotherapeutic drugs. Unfortunately, none has approved for clinical use due to toxicity issues. Some of the P-gp inhibitors tested in the clinics are reported to have cross-reactivity with CYP450 drug-metabolizing enzymes, resulting in unpredictable pharmacokinetics and toxicity of co-administered chemotherapeutic drugs. In this study, two piperine analogs (3 and 4) having lower cross-reactivity with CYP3A4 drug-metabolizing enzyme are identified as P-glycoprotein (P-gp) inhibitors through computational design, followed by synthesis and testing in MDR cancer cell lines over-expressing P-gp (KB ChR 8-5, SW480-VCR, and HCT-15). Both the analogs significantly increased the vincristine efficacy in MDR cancer cell lines at low micromole concentrations. Specifically, 3 caused complete reversal of vincristine resistance in KB ChR 8-5 cells and found to act as competitive inhibitor of P-gp as well as potentiated the vincristine-induced NF-KB-mediated apoptosis. Therefore, 3 ((2E,4E)-1-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-5-(4-hydroxy-3-methoxyphenyl)penta-2,4-dien-1-one) can serve as a potential P-gp inhibitor for in vivo investigations, to reverse multidrug resistance in cancer.

RSK inhibitor BI-D1870 inhibits acute myeloid leukemia cell proliferation by targeting mitotic exit.

The 90 kDa Ribosomal S6 Kinase (RSK) drives cell proliferation and survival in cancers, although its oncogenic mechanism has not been well characterized. Phosphorylated level of RSK (T573) was increased in acute myeloid leukemia (AML) patients and associated with poor survival. To examine the role of RSK in AML, we analyzed apoptosis and the cell cycle profile following treatment with BI-D1870, a potent inhibitor of RSK. BI-D1870 treatment increased the G2/M population and induced apoptosis in AML cell lines and patient AML cells. Characterization of mitotic phases showed that the metaphase/anaphase transition was significantly inhibited by BI-D1870. BI-D1870 treatment impeded the association of activator CDC20 with APC/C, but increased binding of inhibitor MAD2 to CDC20, preventing mitotic exit. Moreover, the inactivation of spindle assembly checkpoint or MAD2 knockdown released cells from BI-D1870-induced metaphase arrest. Therefore, we investigated whether BI-D1870 potentiates the anti-leukemic activity of vincristine by targeting mitotic exit. Combination treatment of BI-D1870 and vincristine synergistically increased mitotic arrest and apoptosis in acute leukemia cells. These data show that BI-D1870 induces apoptosis of AML cells alone and in combination with vincristine through blocking mitotic exit, providing a novel approach to overcoming vincristine resistance in AML cells.

Up-regulation of GLI1 in vincristine-resistant rhabdomyosarcoma and Ewing sarcoma

BackgroundThe clinical significance of GLI1 expression either through canonical Hedgehog signal transduction or through non-canonical mechanisms in rhabdomyosarcoma (RMS) or Ewing sarcoma (EWS) is incompletely understood. We tested a role for Hedgehog (HH) signal transduction and GL11 expression in development of vincristine (VCR) resistance in RMS and EWS.MethodsWe characterized baseline expression and activity of HH pathway components in 5 RMS (RD, Rh18, Ruch-2, Rh30, and Rh41) and 5 EWS (CHLA9, CHLA10, TC32, CHLA258, and TC71) cell lines. We then established VCR-resistant RMS and EWS cell lines by exposing cells to serially increasing concentrations of VCR and determining the IC50. We defined resistance as a ≥ 30-fold increase in IC50 compared with parental cells. We determined changes in gene expression in the VCR-resistant cells compared with parental cells using an 86-gene cancer drug resistance array that included GLI1 and tested the effect of GLI1 inhibition with GANT61 or GLI1 siRNA on VCR resistance.ResultsWe found evidence for HH pathway activity and GLI1 expression in RMS and EWS cell lines at baseline, and evidence that GLI1 contributes to survival and proliferation of these sarcoma cells. We were able to establish 4 VCR-resistant cell lines (Ruch-2VR, Rh30VR, Rh41VR, and TC71VR). GLI1 was significantly up-regulated in the Rh30VR, Rh41VR, and TC71VR cells. The only other gene in the drug resistance panel that was significantly up-regulated in each of these VCR-resistant cell lines compared with their corresponding parental cells was the GLI1 direct target and multidrug resistance gene, ATP-binding cassette sub-family B member 1 (MDR1). We established major vault protein (MVP), which was up-regulated in both vincristine-resistant alveolar RMS cell lines (Rh30VR and Rh41VR), as another direct target of GLI1 during development of drug resistance. Treatment of the VCR-resistant cell lines with the small molecule inhibitor GANT61 or GLI1 siRNA together with VCR significantly decreased cell viability at doses that did not reduce viability individually.ConclusionsThese experiments demonstrate that GLI1 up-regulation contributes to VCR resistance in RMS and EWS cell lines and suggest that targeting GLI1 may benefit patients with RMS or EWS by reducing multidrug resistance.

Destruction of a Microtubule-Bound MYC Reservoir during Mitosis Contributes to Vincristine's Anticancer Activity.

Tightly regulated activity of the transcription factor MYC is essential for orderly cell proliferation. Upon deregulation, MYC elicits and promotes cancer progression. Proteasomal degradation is an essential element of MYC regulation, initiated by phosphorylation at Serine62 (Ser62) of the MB1 region. Here, we found that Ser62 phosphorylation peaks in mitosis, but that a fraction of nonphosphorylated MYC binds to the microtubules of the mitotic spindle. Consequently, the microtubule-destabilizing drug vincristine decreases wild-type MYC stability, whereas phosphorylation-deficient MYC is more stable, contributing to vincristine resistance and induction of polyploidy. PI3K inhibition attenuates postmitotic MYC formation and augments the cytotoxic effect of vincristine. IMPLICATIONS: The spindle's function as a docking site for MYC during mitosis may constitute a window of specific vulnerability to be exploited for cancer treatment.

C-Myc inhibition sensitizes pre-B ALL cells to the anti-tumor effect of vincristine by altering apoptosis and autophagy: Proposing a probable mechanism of action for 10058-F4

Unlike the broad spectrum efficacies in wiping the malignant cells out, application of vincristine (VCR) in acute lymphoblastic leukemia (ALL) therapeutic protocol is partially restricted due to its high frequent resistant rate. Although several mechanisms have been enumerated for VCR resistance, to the best of our knowledge, there is no report reflecting the suppressive effect of oncogenic pathways on VCR cytotoxicity in ALL. The results of the present study indicated that both pre-B ALL-derived REH and Nalm-6 cells were partly resistant to VCR, with this note that Nalm-6 cells displayed more resistant phenotype. More interestingly, we showed for the first time that among inhibitors of different signaling pathways including those targeting PI3K, ERK, and NF-κB, the enhancive effect of small molecule inhibitor of c-Myc 10058-F4 was more significant on VCR cytotoxicity. Inhibition of c-Myc in VCR-treated Nalm-6 cells promoted a caspase-3-dependent apoptosis not only through altering the balance between death promoters to death suppressors, but also via modulating the expression of autophagy-related genes. Noteworthy, favorable impact of 10058-F4 on VCR anti-leukemic effect was not restricted to the induction of cell death and this agent also reinforced VCR anti-proliferative effect through disturbing cell cycle progression and hampering the expression of Pin1 and hTERT. In conclusion, it seems that targeting c-Myc could produce a synergistic anti-cancer effect with VCR and provide a fundamental infrastructure for a promising approach in ALL.

Secreted amphiregulin promotes vincristine resistance in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the most common type of oral cancer. Despite advances in surgery, radiotherapy and chemotherapy, the overall 5‑year survival rate of patients with OSCC has not significantly improved. In addition, the prognosis of patients with advanced‑stage OSCC remains poor. Therefore, it is necessary to develop novel therapeutic modalities. Vincristine (VCR), a naturally occurring vinca alkaloid, is a classical microtubule‑destabilizing agent and is widely used in the treatment of a number of cancers. Despite the proven antitumor benefits of VCR treatment, one of the major reasons for the failure of treatment is drug resistance. Changes in the tumor microenvironment are responsible for cross‑talk between cells, which may facilitate drug resistance in cancers; secreted proteins may promote communication between cancer cells to induce the development of resistance. To identify the secreted proteins involved in VCR resistance, conditioned media was obtained, and an antibody array was conducted to screen a comprehensive secretion profile between VCR‑resistant (SAS‑VCR) and parental (SAS) OSCC cell lines. The results showed that amphiregulin (AREG) was highly expressed and secreted in SAS‑VCR cells. Pretreatment with exogenous recombinant AREG markedly increased drug resistance against VCR in OSCC cells, as assessed by an MTT assay. Colony formation, MTT and western blot assays were performed to investigate the effects of AREG knockdown on VCR sensitivity. The results indicated that AREG expression can regulate VCR resistance in OSCC cells; overexpression of AREG increased VCR resistance in parental cells, whereas AREG knockdown decreased the VCR resistance of resistant cells. In addition, it was also demonstrated that the glycogen synthase kinase‑3β pathway may be involved in AREG‑induced VCR resistance. These findings may provide rationale to combine VCR with blockade of AREG‑related pathways for the effective treatment of OSCC.

Overexpression of heme oxygenase-1 in microenvironment mediates vincristine resistance of B-cell acute lymphoblastic leukemia by promoting vascular endothelial growth factor secretion.

Chemoresistance often causes treatment failure of B-cell acute lymphoblastic leukemia (B-ALL). However, the mechanism remains unclear at present. Herein, overexpression of heme oxygenase-1 (HO-1) was found in the bone marrow stromal cells (BMSCs) from B-ALL patients developing resistance to vincristine (VCR), a chemotherapeutic agent. Two B-ALL cell lines Super B15 and CCRF-SB were cocultured with BMSCs transfected with lentivirus to regulate the expression of HO-1. Silencing HO-1 expression in BMSCs increased the apoptotic rates of B-ALL cell lines induced by VCR, whereas upregulating HO-1 expression reduced the rate. Cell cycle can be arrested in the G2/M phase by VCR. In contrast, B-ALL cells were arrested in the G0/G1 phase due to HO-1 overexpression in BMSCs, which avoided damage from the G2/M phase. Vascular endothelial growth factor (VEGF) in BMSCs, as a key factor in the microenvironment-associated chemoresistance, was also positively coexpressed with HO-1. VEGF secretion was markedly increased in BMSCs with HO-1 upregulation but decreased in BMSCs with HO-1 silencing. B-ALL cell lines became resistant to VCR when cultured with VEGF recombinant protein, so VEGF secretion induced by HO-1 expression may promote the VCR resistance of B-ALL cells. As to the molecular mechanism, the PI3K/AKT pathway mediated regulation of VEGF by HO-1. In conclusion, this study clarifies a mechanism by which B-ALL is induced to resist VCR through HO-1 overexpression in BMSCs, and provides a novel strategy for overcoming VCR resistance in clinical practice.

MicroRNA-155 controls vincristine sensitivity and predicts superior clinical outcome in diffuse large B-cell lymphoma

AbstractA major clinical challenge of diffuse large B-cell lymphoma (DLBCL) is that up to 40% of patients have refractory disease or relapse after initial response to therapy as a result of drug-specific molecular resistance. The purpose of the present study was to investigate microRNA (miRNA) involvement in vincristine resistance in DLBCL, which was pursued by functional in vitro analysis in DLBCL cell lines and by outcome analysis of patients with DLBCL treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). Differential miRNA expression analysis identified miR-155 as highly expressed in vincristine-sensitive DLBCL cell lines compared with resistant ones. Ectopic upregulation of miR-155 sensitized germinal-center B-cell-like (GCB)–DLBCL cell lines to vincristine, and consistently, reduction and knockout of miR-155 induced vincristine resistance, documenting that miR-155 functionally induces vincristine sensitivity. Target gene analysis identified miR-155 as inversely correlated with Wee1, supporting Wee1 as a target of miR-155 in DLBCL. Chemical inhibition of Wee1 sensitized GCB cells to vincristine, suggesting that miR-155 controls vincristine response through Wee1. Outcome analysis in clinical cohorts of DLBCL revealed that high miR-155 expression level was significantly associated with superior survival for R-CHOP-treated patients of the GCB subclass, independent of international prognostic index, challenging the commonly accepted perception of miR-155 as an oncomiR. However, miR-155 did not provide prognostic information when analyzing the entire DLBCL cohort or activated B-cell–like classified patients. In conclusion, we experimentally confirmed a direct link between high miR-155 expression and vincristine sensitivity in DLBCL and documented an improved clinical outcome of GCB-classified patients with high miR-155 expression level.

Oncogenic PITX2 facilitates tumor cell drug resistance by inverse regulation of hOCT3/SLC22A3 and ABC drug transporters in colon and kidney cancers

Oncogenic pituitary homeobox 2 (PITX2), a de facto master regulator of developmental organ asymmetry, previously upregulated multidrug resistance (MDR) P-glycoprotein ABCB1 in A498 renal cell carcinoma (RCC) cells. The role of PITX2 isoforms in MDR cancers was investigated. Data mining correlated elevated PITX2 in >30% of cancers analyzed, maximally in colon (4.4-fold), confirmed in co-immunostaining of colon and renal cancer microarrays wherein ABCB1 concomitantly increased in RCC. Drug-resistant colorectal adenocarcinoma Colo320DM cells exhibited increased nuclear PITX2 (40-fold), PITX2 promoter activity (27-fold) and ABCB1 (8000-fold) compared to drug-sensitive Colo205. ABCB1 inhibitor PSC833/valspodar or PITX2 siRNA reversed doxorubicin resistance. Nuclei from Colo320DM and A498 cells harbored PITX2A/B1 and PITX2A/B1/B2/Cα/Cβ, respectively. ChIP-qPCR evidenced PITX2 promoter binding in drug exporters ABCB1, ABCC1, ABCG2 and importer hOCT3/SLC22A3. In A498, 786-O, Caki-1, Colo320DM, and Caco2 cells, PITX2 siRNA diminished exporters, increased hOCT3/SLC22A3 expression and activity, and reverted vincristine resistance. Heterologous PITX2 expression induced ABCB1, repressed hOCT3/SLC22A3, enhanced vincristine resistance and diminished proliferation inhibition wherein PITX2A and PITX2C were most effective. Furthermore, PITX2 activity and MDR depended on phosphorylation by GSK3 in A498 cells. Conclusively, oncogenic PITX2 limits sensitizing drug uptake and potentiates cytoprotective drug efflux, contributing to MDR phenotype.

Retracted Article: Long non-coding RNA GACAT1 alleviates doxorubicin and vincristine resistance through a PTEN/AKT/mTOR/S6K1 regulatory pathway in gastric cancer.

Gastric cancer (GC) is a major global health problem. Chemotherapy is a common therapeutic strategy for cancers including GC. However, chemoresistance strikingly limits the clinical applications of chemotherapeutic drugs. Long non-coding RNAs (lncRNAs) have been widely reported to be implicated in the pathogenesis and chemoresistance of cancers including GC. Our work aims to investigate the roles and molecular mechanisms of lncRNA gastric cancer-associated transcript 1 (GACAT1) in regulating doxorubicin (ADR) and vincristine (VCR) resistance in GC. In this text, RT-qPCR assay showed that GACAT1 expression was markedly reduced in ADR- or VCR-resistant GC (SGC7901/ADR or SGC7901/VCR) cells and GC tissues. CCK-8 assay and flow cytometry analysis revealed that GACAT1 overexpression alleviated the resistance of GC cells to ADR and VCR. RT-qPCR and western blot assay disclosed that GACAT1 deactivated the AKT/mTOR/S6K1 signaling pathway and promoted PTEN expression in SGC7901/ADR or SGC7901/VCR cells. Restoration experiments demonstrated that GACAT1 attenuated ADR or VCR resistance by regulating the PTEN/AKT/mTOR/S6K1 pathway in SGC7901/ADR or SGC7901/VCR cells. In vivo experiments demonstrated that GACAT1 overexpression inhibited tumor growth and enhanced ADR- or VCR-mediated anti-tumor effects in GC xenograft tumor models. Taken together, these data revealed that GACAT1 weakened the resistance of GC cells to ADR and VCR by the PTEN/AKT/mTOR/S6K1 regulatory pathway in vitro and in vivo, shedding new light on GACAT1 upregulation as a potential strategy to alleviate chemoresistance in GC.

Downregulation of DBN1 is related to vincristine resistance in colon cancer cells.

This study was aimed to investigate the relationship between the expression of drebrin (DBN1) gene and resistance in colon cancer to reveal the mechanism of tumor drug resistance and provide a basis for the reversal of this drug resistance in tumor cells. The human colon carcinoma cell line HCT-8 was used, and vincristine (VCR)-resistant colon cancer cell line HCT-8/V was established by gradually increasing the concentration of VCR. Polymerase chain reaction (PCR) primers were designed for DBN1 gene. The DBN1 differential expression in colon cancer sensitive and resistant cell lines was detected by fluorescence quantitative PCR. Western blot analysis was used to study DBN1 expression in the resistant cells further. VCR resistance of HCT-8/V cell line was established. Quantitative PCR and Western blot results showed that DBN1 expression in the resistant cell line was significantly lower, the difference being statistically significant (P < 0.05). Low DBN1 gene expression may be associated with colon cancer cell resistance to VCR.

A Novel PAX5-KIDINS220 Fusion Protein Activates Multiple Signals Associated with Therapeutic Resistance

Background: It is well known that PAX5 related fusion proteins are mainly associated with leukemogenesis of B cell precursor acute lymphoblastic leukemia (B-ALL) through dominant negative effect against normal PAX5, resulting in impairment of B cell differentiation. However, different biological property of PAX5 fusion protein which is related to B-ALL has not been fully investigated. Here, we performed functional analysis of novel fusion protein, PAX5-KIDINS220 (P-K220) which was identified in pediatric Ph-like ALL patient. Methods: Full length of P-K220 fusion gene was cloned into pRetroX-Tight-Pur retroviral vector. We also established the PAX5-N construct that contained only PAX5 region (1-306 aa) of P-K220 so that we could analyze the importance of KIDINS220 in P-K220. Then, Ba/F3 cells, which are IL-3 dependent murine pro B-ALL cells, were transduced with this retroviral vector to establish Ba/F3 cells expressing P-K220 or PAX5-N under doxycycline (DOX) dependent manner. P-K220 was also cloned into pAcGFP1-C1 vector and pMSCVneo vector to perform localization assay and luciferase reporter assay. Gene expression analysis was performed using Mouse Genome 430 2.0 Array. In cytotoxic assay using co-culture system with murine mesenchymal stromal cell, MS-5, Ba/F3 cells that were pre-stained with Carboxyfluorescein diacetate succinimidyl ester (CFSE) were seeded on MS-5 stromal cell, then 10 nM vincristine (VCR) was applied. 72 hours later, flow cytometric analysis was performed to determine the fraction and proliferation ability of viable Ba/F3 cells. The proliferation ability was estimated by CFSE fluorescence that attenuates depending on cell proliferation. Results and discussions: P-K220 and PAX5-N were successfully expressed in Ba/F3 cells, confirmed by western blotting. Localization assay revealed that GFP tagged P-K220 was localized in a nucleus of HEK293-T cells under confocal microscopy, suggesting that P-K220 acted as a transcriptional factor. Luciferase reporter assay revealed that P-K220 protein inhibited PAX5 transcriptional activity in dominant negative fashion. Although P-K220 was identified in Ph-like ALL patient and activated JAK2-STAT5 pathway through reduction of Socs5 expression, Ba/F3 cells expressing P-K220 protein did not acquire IL-3 independency. P-K220 attenuated proliferation of Ba/F3 cells (p=0.02), which was in contrast to PAX5-JAK2. To reveal which pathways were affected by P-K220, gene expression analysis was performed. Gene set enrichment analysis revealed that multiple pathways related to chemotaxis, migration, such as IL-15 pathway, were activated in P-K220 expressing Ba/F3 cells. RQ-PCR and western blotting confirmed P-K220 induced expression of IL-15 in RNA (p=0.018) and protein level, suggesting that P-K220 might be associated with infiltration of leukemic cells into extramedullary site. Next, we examined whether P-K220 was associated with sensitivity of chemotherapeutic agents, such as vincristine (VCR), cytarabine, prednisolone, methotrexate, and 6-mercaptoprine. P-K220 expressing Ba/F3 cells showed higher VCR IC50 (0.4 nM vs 2.5 nM) and decreased Annexin V positive fraction under the condition of 10 nM VCR (39.4 % vs 23.8 %, p=0.04). In co-culture analysis with MS-5 stromal cell under the 10 nM VCR, the fraction of viable Ba/F3 cells that adhered to MS-5 was significantly increased with expression of P-K220 than PAX5-N or Dox (-) (11.6% vs 7.3% vs 6.8%, p<0.001), and proliferation ability was also significantly increased with expression of P-K220, that was estimated by mean CFSE fluorescence (479.6 vs 990.0 vs 909.7, p<0.001), suggesting P-K220 was associated with VCR resistance. Gene expression analysis and RQ-PCR revealed that P-K220 induced expression of Abcc5 which was reported to be associated with VCR resistance (p=0.003). Conclusion: P-K220 did not induce high proliferation of Ba/F3 cells even though identified in Ph-like ALL patient. However, P-K220 might be associated with treatment resistance through the infiltration of leukemic cells into extramedullary sites partly via activation of IL-15 pathway and resistance to VCR via induction of Abcc5. DisclosuresNo relevant conflicts of interest to declare.

Survivin expression modulates the sensitivity of A549 lung cancer cells resistance to vincristine.

Lung cancer is the leading cause of cancer-associated mortalities worldwide. Chemotherapeutic drug vincristine is widely used to treat lung cancer; however, the acquisition of drug resistance is the major limitation of chemotherapy, and it is thus important to determine the mechanism underlying vincristine resistance in lung cancer. Survivin has been reported to be associated with the development of drug resistance and be involved in the progression of non-small cell lung cancer. In the present study, a vincristine-resistant lung cancer cell line, A549/VCR, was used to investigate the possible involvement of survivin in the acquisition of vincristine resistance. Western blot analysis demonstrated that survivin protein expression level was markedly higher in A549/VCR cells compared with in control A549 cells, whereas p53 expression level was lower in A549/VCR cells compared with in A549 cells. Thus, wild-type p53 was overexpressed in A549/VCR cells and it reversed vincristine resistance of A549/VCR cells via the inhibition of survivin expression. Furthermore, survivin was knocked down by small interfering RNA technology and the effects on viability and apoptosis of resistant cells were investigated. MTT, Annexin V-fluorescein isothiocyanate/propidium iodide and caspase-3 activity assays indicated that survivin silencing significantly inhibited cell viability and enhanced apoptosis induced by vincristine treatment in A549/VCR cells compared with non-silenced A549/VCR cells. These results suggested that survivin expression regulated by p53 may serve an important role in drug resistance in A549/VCR cells and may be a potential target for enhancing vincristine sensitivity in A549 lung cancer cells. Additionally, the present study revealed that A549/VCR cells exhibited cross resistance to methotrexate (MTX) and survivin silencing re-sensitized A549/VCR cells to MTX, indicating the crucial role of survivin in regulating A549 cells sensitivity to anticancer drugs. The results of the present study are significant for determining the underlying mechanism of vincristine resistance in lung cancer.

Reduced Lipocalin 2 Expression Contributes to Vincristine Resistance in Human Colon Cancer Cells.

Vincristine (VCR) resistance can lead to cancer chemotherapy failure. Although changes in gene expression are responsible for drug resistance, the specific identities and roles of these genes remain unclear. In this study, we aimed to identify differentially expressed genes and mechanisms of VCR resistance in colorectal cancer (CRC) cells. A VCR-resistant CRC cell line (HCT-8/VCR) was established, and differentially expressed proteins between HCT-8 and HCT-8/VCR cells were screened using a human cytokine array; the results were confirmed by reverse transcription polymerase chain reaction and Western blotting. Furthermore, differentially expressed proteins were downregulated using siRNA, and cell proliferation and apoptosis were assessed by Cell Counting Kit-8 assay and flow cytometry, respectively. Compared with HCT-8 CRC cells, HCT-8/VCR cells showed downregulation of lipocalin 2 (LCN2). We found that siRNA-mediated downregulation of LCN2 in HCT-8 cells significantly increased VCR resistance. Furthermore, when we downregulated LCN2, we observed significant decreases in apoptosis, but no significant effect on cell cycle. Overall, these results demonstrate that LCN2 plays an important role in VCR resistance and is a potential therapeutic target for this disease.