The positive correlation between drug addiction and drug dosage in vemurafenib-resistant melanoma cells is underpinned by activation of ERK1/2-FRA-1 pathway.

Malignant melanoma is a kind of highly invasive and deadly diseases. The BRAF inhibitor (BRAFi) such as vemurafenib could achieve a high response rate in melanoma patients with BRAF mutation. However, melanoma cells could easily develop resistance as well as addiction to BRAFi. Based on the drug addiction, intermittent treatment has been proposed to select against BRAFi-resistant melanoma cells. Because different dosages of BRAFi might be used in patients, it is necessary to know about the relationship between drug dosage and the degree of addiction. To address the problem, four drug-resistant melanoma cell sublines (A375/R0.5, A375/R2.0, M14/R0.5 and M14/R2.0) were established by continuously exposure of melanoma A375 or M14 cells to 0.5 or 2.0 µM vemurafenib. Vemurafenib withdrawal resulted in much stronger suppression on clone formation in A375/R2.0 and M14/R2.0, compared with A375/R0.5 and M14/R0.5, respectively. Meanwhile, stronger upregulation of ERK1/2-FRA-1 pathway could be observed in A375/R2.0 and M14/R2.0. Further detection showed that some proinflammatory cytokines downstream of ERK1/2-FRA-1 pathway were upregulated after drug withdrawal, and the conditioned medium collected from the resistant A375 cells could inhibit clone formation. Furthermore, vemurafenib withdrawal resulted in suppressed cell proliferation rather than cell senescence, with stronger effect on A375/R2.0 compared with A375/R0.5. This study suggested that the depth of vemurafenib addiction in resistant melanoma cells is positively correlated to the drug dosage, which might be underpinned by the ERK1/2-FRA-1 pathway and the related cytokines.

IL-1α inhibits proliferation and adipogenic differentiation of human adipose-derived mesenchymal stem cells through NF-κB- and ERK1/2-mediated proinflammatory cytokines.

Dysfunctional adipogenesis such as subcutaneous lipoatrophy is closely related to insulin resistance and metabolic disorders. Although the expression or release of the cytokine interleukin-1α (IL-1α) is known to increase in adipose tissue in response to cell death, cell senescence, aging, or solar radiation, the regulatory role of IL-1α in adipogenesis has not been sufficiently investigated. To investigate the problem, we explored the effect of IL-1α on the proliferation and adipogenic differentiation of human adipose-derived mesenchymal stem cells (ADSCs) using cell counting, alamarBlue assay, oil red O staining, Western blot, among others. The results showed that IL-1α evidently inhibited the proliferation and adipogenic differentiation of ADSCs, which might be related with the activated nuclear factor-κB (NF-κB) and extracellular signal-regulated kinase (ERK) 1/2 pathways. Early-stage adipogenic differentiation was more sensitive to IL-1α than late-stage differentiation. After differentiation of ADSCs into mature adipocytes, adding of IL-1α had no obvious influence on the cellular morphology, including lipid droplet accumulation. IL-1α enhanced the expression of proinflammatory cytokines, such as IL-8, IL-6, CCL2 (C-C motif chemokine ligand 2), and IL-1ß, when added into the adipogenic medium of ADSCs. Blocking IL-8 and IL-6 with neutralizing antibodies partially alleviated the inhibitory effect of IL-1α on the proliferation and adipogenic differentiation. The results suggest that IL-1α inhibits adipogenesis through activation of NF-κB and ERK1/2 pathways and subsequent upregulation of proinflammatory cytokines in ADSCs. IL-1α might play an important role in mediating lipoatrophy by regulation of ADSCs.

[Induction of robust senescence-associated secretory phenotype in mouse NIH-3T3 cells by mitomycin C].

Senescence-associated secretory phenotype (SASP) is often a concomitant result of cell senescence, embodied by the enhanced function of secretion. The SASP factors secreted by senescent cells include cytokines, proteases and chemokines, etc, which can exert great influence on local as well as systemic environment and participate in the process of cell senescence, immunoregulation, angiogenesis, cell proliferation and tumor invasion, etc. Relative to the abundance of SASP models in human cells, the in vitro SASP model derived from mouse cells is scarce at present. Therefore, the study aimed to establish a mouse SASP model to facilitate the research in the field. With this objective, we treated the INK4a-deficient mouse NIH-3T3 cells and the wildtype mouse embryonic fibroblasts (MEF) respectively with mitomycin C (MMC), an anticarcinoma drug which could induce DNA damage. The occurring of cell senescence was evaluated by cell morphology, ß-gal staining, integration ratio of EdU and Western blot. Quantitative RT-PCR and ELISA were used to detect the expression and secretion of SASP factors, respectively. The results showed that, 8 days after the treatment of NIH-3T3 cells with MMC (1 µg/mL) for 12 h or 24 h, the cells became enlarged and the ratios of ß-gal-positive (blue-stained) cells significantly increased, up to 77.4% and 90.4%, respectively. Meanwhile, the expression of P21 protein increased and the integration ratios of EdU significantly decreased (P < 0.01). Quantitative RT-PCR detection showed that the mRNA levels of several SASP genes, including IL-6, TNF-α, IL-1α and IL-1ß increased evidently. ELISA detection further observed an enhanced secretion of IL-6 (P < 0.01). On the contrary, although wildtype MEF could also be induced into senescence by MMC treatment for 12 h or 24 h, embodied by the enlarged cell volume, increased ratios of ß-gal-positive cells (up to 71.7% and 80.2%, respectively) and enhanced expression of P21 protein, the secretion of IL-6 displayed no significant change. Our study indicated that, although MMC could induce senescence in both mouse NIH-3T3 cells and wildtype MEF, only senescent NIH-3T3 cells displayed the canonical SASP phenomena. Current study suggested that senescent NIH-3T3 cells might be an appropriate in vitro SASP model of mouse cells.

LHPP Inhibits the Proliferation and Metastasis of Renal Cell Carcinoma.

Renal cell carcinoma (RCC) is one of the ten most common cancers in the globe. Despite the diagnosis and treatment of renal cell carcinoma that have made great improvements, the morbidity and mortality rates of renal cell carcinoma remain unchanged remarkably. LHPP is a kind of histidine phosphatases, acting as a tumor suppressor in the progression of various cancers. In this study, we found that LHPP was significantly downregulated in RCC tissues and cell lines. Decreased expression of LHPP was closely correlated with tumor size and postoperative metastasis of RCC patients. In addition, overexpression of LHPP inhibited the proliferation and metastasis of RCC. However, suppression of LHPP promoted the proliferation and metastasis of RCC. In conclusion, our results presented the important role of LHPP in the development and progression of RCC.

Senescence-associated secretory factors induced by cisplatin in melanoma cells promote non-senescent melanoma cell growth through activation of the ERK1/2-RSK1 pathway.

Although targeted therapy and immunotherapy greatly improve the outcome of melanoma, drug resistance and low response rates still maintain the unsubstitutability of traditional chemotherapy. Cisplatin (CDDP) is widely used in different types of tumours with high response rates, but it generally has low efficiency in melanoma. The mechanisms underpinning the phenomena are not sufficiently understood. Here we demonstrated that various melanoma cell lines adopted senescence phenotype after CDDP treatment in contrast to the other types of tumour cells. CDDP treatment induced melanoma A375 cells into senescence through the sequential activation of the DNA damage response and the P53/P21 pathway. All the senescent melanoma cells induced by CDDP alone or the combination of CDDP and dacarbazine developed robust senescence-associated secretory phenotype (SASP), that is, the secretion of multiple cytokines. IL-1α was an early component and an upstream regulator of SASP. Similarly, CDDP either alone or combined with dacarbazine could induce melanoma cell senescence and SASP in either A375 or B16F10 melanoma xenograft mice. The supernatant of senescent A375 cells promoted the growth of normal non-senescent A375 cells and enhanced their expression and secretion of IL-8 through the activation of the ERK1/2-RSK1 pathway. The transplantation of non-senescent and senescent A375 cells together into nude mice showed accelerated tumour growth compared with transplanting non-senescent cells alone; no tumours developed when transplanting senescent cells alone. Following CDDP administration in A375-bearing mice, the intratumour injection of neutralisation antibodies targeting the SASP factors IL-1α or IL-8 evidently delayed tumour growth. The results suggest that the CDDP-induced senescent melanoma cells promote non-senescent cells proliferation through the activation of ERK1/2-RSK1 pathway by the SASP factors. Cell senescence and concomitant SASP may be the particular mechanisms for melanoma to resist chemotherapeutics.