Growth Inhibition Of Melanoma Cells Research Articles

Pseudopodium enriched atypical kinase 1(PEAK1) promotes invasion and of melanoma cells by activating JAK/STAT3 signals

ABSTRACT Pseudopodium enriched atypical kinase 1(PEAK1) is a non-receptor tyrosine kinase, which is enriched in the pseudopodia of migrating cells and plays an important role in regulating cell migration and proliferation. In the study, we investigate the therapeutic effect of PEAK1 on melanoma cells in vitro and in vivo. We used a lentiviral vector to express short hairpin RNAs (Lv-PEAK1 shRNA) for inhibiting PEAK1 expression in the melanoma SKMEL28 cells. A full-length PEAK1 gene was cloned into the pcDNA 3.1 (+) plasmid and used to infect the melanoma SKMEL19 cells. P6 (also known as Pyridines 6, EMD Chemicals), the Pan-JAK inhibitor, was used to inhibit the Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway. The cell counting kit-8 (CCK-8), colony formation assay and transwell assay were used to detect cell proliferation, growth and invasion in vitro. The effect of PEAK1 on melanoma progression in vivo was also evaluated. Protein expression of PEAK1, E-cadherin, vimentin and JAK/STAT3 was measured using western blot assay or immunohistochemistry. The results showed that enforced PEAK1 expression facilitated melanoma cell growth, invasion and metastasis via activating JAK/STAT3 signals, and PEAK1 knockdown inhibited melanoma cell growth, invasion and metastasis via inactivating JAK/STAT3 signals. Further work demonstrated that P6 (500 nM) treatment reversed PEAK1-induced effect in melanoma cells. PEAK1 promotes tumorigenesis and metastasis via activating JAK/STAT3 signals, and PEAK1 knockdown reduced tumorigenesis and metastasis in melanoma via inactivating JAK/STAT3 signals, providing a novel therapeutic strategy for melanoma treatment.

Baicalein and Baicalin Promote Melanoma Apoptosis and Senescence via Metabolic Inhibition.

Malignant melanoma is one of the most common and dangerous skin cancers with a high rate of death every year. Furthermore, N-RAS and B-RAF mutations in melanoma cells increase the difficulties for clinical treatment in patients. Therefore, development of effective and universal drugs against melanoma is urgently needed. Here we demonstrate that baicalein and baicalin, the active components of the Chinese traditional medicinal plant Scutellaria baicalensis Georgi, can significantly inhibit melanoma cell growth and proliferation, suppress tumor cell colony formation and migration, as well as induce apoptosis and senescence in melanoma cells. The anti-tumor effects mediated by baicalein and baicalin are independent of N-RAS and B-RAF mutation statuses in melanoma cells. Mechanistically, we identify that the suppression of baicalein and baicalin on melanoma cells is due to inhibition of tumor cell glucose uptake and metabolism by affecting the mTOR-HIF-1α signaling pathway. In addition, we demonstrated that baicalein and baicalin can suppress tumorigenesis and tumor growth in vivo in the melanoma model. These studies clearly indicate that baicalein and baicalin can control tumor growth and development metabolically and have great potential as novel and universal drugs for melanoma therapy.

Nuciferine Inhibits Skin Cutaneous Melanoma Cell Growth by Suppressing TLR4/NF-κB Signaling.

Melanoma causes more than 80% of deaths from all dermatologic cancers. Hence, screening and identifying effective compounds to inhibit the growth of melanoma have crucial importance in basic and clinical treatment. High throughput screening was performed to screen and identify compounds that have anti-melanoma ability. Melanoma cell and mouse allograft models were used to examine the anti-tumor effects of Nuciferine (NCFR). Western blot, qPCR, and lentivirus overexpression were applied to detect the activation of the TLR4/NF-κB signaling pathway. NCFR administration significantly suppressed melanoma cell growth and tumor size by inhibiting the phosphorylation of p65. NCFR treatment also could suppress TNF-α-induced activation of NF-κB signaling. The anti-tumor effect of NCFR might be mediated by targeting Toll-like receptors 4. NCFR inhibits melanoma cell growth and suppresses tumor size, which provides potential therapeutic strategies for melanoma treatment.

Long Noncoding RNA CCAT1 Functions as a Competing Endogenous RNA to Upregulate ITGA9 by Sponging MiR-296-3p in Melanoma.

BackgroundMelanoma is aggressive and lethal melanocytic neoplasm, and its incidence has increased worldwide in recent decades. Accumulating evidence has showed that various long noncoding RNAs (lncRNAs) participated in occurrence of malignant tumors, including melanoma. The present study was designed to investigate function of lncRNA colon cancer-associated transcript-1 (CCAT1) in melanoma.MethodsThe expression levels of CCAT1, miR-296-3p and Integrin alpha9 (ITGA9) in melanoma tissues or cells were measured using real-time quantitative polymerase chain reaction (RT-qPCR). The concentrations of glucose and lactate were measured for assessing glycolysis of melanoma cells. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazol-3-ium bromide (MTT), flow cytometry, and transwell assays were conducted to assess proliferation, apoptosis, and migration of melanoma cells. Western blot assay was performed to measure the protein expression of ITGA9, hexokinase 2 (HK2), and epithelial–mesenchymal transition (EMT)-related proteins in melanoma tissues or cells. The relationship among CCAT1, miR-296-3p, and ITGA9 was predicted and confirmed by bioinformatics analysis, dual-luciferase reporter, and RNA immunoprecipitation (RIP) assay, respectively. A xenograft experiment was established to assess the effect of CCAT1 knockdown in vivo.ResultsCCAT1 was effectively increased in melanoma tissues and cells compared with matched controls, and deficiency of CCAT1 impeded cell glycolysis, proliferation, migration while induced apoptosis, which were abrogated by knockdown of miR-296-3p in melanoma cells. In addition, our findings revealed that ITGA9 overexpression abolished miR-296-3p overexpression-induced effects on melanoma cells. Importantly, CCAT1 regulated ITGA9 expression by sponging miR-296-3p. The results of xenograft experiment suggested that CCAT1 silencing inhibited melanoma cell growth in vivo.ConclusionLncRNA CCAT1 promoted ITGA9 expression by sponging miR-296-3p in melanoma.

Luteolin inhibits melanoma growth in vitro and in vivo via regulating ECM and oncogenic pathways but not ROS

Luteolin inhibited growth of several cancer cells in vitro in previous studies, with limited in vivo studies, and no comprehensive understanding of molecular mechanisms at genomics level. This study identified luteolin as an effective agent to inhibit melanoma cell growth in vitro and in vivo. Molecular studies and genomic profiling were used to identify the mechanism of action of luteolin in melanoma cells. As a ROS (reactive oxygen species) scavenger, luteolin unexpectedly induced ROS; but co-treatment with antioxidants NAC or mito-TEMPO did not rescue cell growth inhibition, although the levels of ROS levels were reduced. Next, we profiled luteolin-induced differentially expressed genes (DEGs) in 4 melanoma cell lines using RNA-Seq, and performed pathway analysis using a combination of bioinformatics software including PharmetRx which was especially effective in discovering pharmacological pathways for potential drugs. Our results show that luteolin induces changes in three main aspects: the cell–cell interacting pathway (extracellular matrix, ECM), the oncogenic pathway and the immune response signaling pathway. Based on these results, we further validated that luteolin was especially effective in inhibiting cell proliferation when cells were seeded at low density, concomitantly with down-regulation of fibronectin accumulation. In conclusion, through extensive DEG profiling in a total of 4 melanoma cell lines, we found that luteolin-mediated growth inhibition in melanoma cells was perhaps not through ROS induction, but likely through simultaneously acting on multiple pathways including the ECM (extracellular matrix) pathway, the oncogenic signaling and the immune response pathways. Further investigations on the mechanisms of this promising compound are warranted and likely result in application to cancer patients as its safety pharmacology has been validated in autism patients.

SUN-LB27 Interleukin-8 - a Possible Target for Melanoma Treatment? In-Vitro Studies Based on Human Melanoma Cell Models

Previous clinical studies showed that menstruating females were better protected in melanoma than post-menopausal women and men of any age. In addition, epidemiological studies showed an increased male mortality in melanoma. But these studies did not correlate with steroid status in females. Our in-vitro study showed female sex hormone progesterone significantly inhibited human melanoma cell growth. Further in-vitro study showed that progesterone action was mediated by a specific suppression of pro-inflammatory cytokine IL-8. Our research also showed that addition of IL-8 (1 ng/ml) to melanoma cells stimulated cell growth (117%) and suppression of IL-8 by curcumin (100 μM) pre-treatment suppressed human melanoma cell growth (26%) in-vitro. This observation prompted us to check the effect of male sex hormones androstenedione (AD) and testosterone (T) on melanoma cell growth. AD and T also suppressed cell growth and IL-8 secretion, but not as significantly as that of progesterone. However, addition of progesterone (10 μM) along with androgens showed an additive effect on the inhibition of melanoma cell growth and suppression of IL-8 secretion. As steroids (P, AD, T) targeted IL-8 for their action, it was decided to check whether vitamin-D3 also targeted IL-8 secretion and cell growth. Active form of vit-D3 (25 μM) also suppressed IL-8 secretion and cell growth. But, addition of progesterone (50 μM) along with D3 significantly suppressed cell growth and IL-8 secretion. This brought IL-8 into focus as a key molecule regulating melanoma cell growth. In order to check whether IL-8 was the molecule involved in regulating melanoma cell growth, IL-8 rescue experiment after curcumin (25 μM) pre-treatment was carried out. IL-8 (100 ng/ml) was able to rescue cell growth completely after pre-treatment with curcumin, suggesting IL-8 was the molecule involved in regulating melanoma cell growth. Literature also suggested important role for IL-8 in regulating melanoma cell growth. Conditional expression of IL-8 in nude mouse by Dr. Singh et al., indicated in-vivo role of IL-8 in melanoma growth and metastasis. Conclusion: Both, in-vitro and in-vivo studies suggested an important role for IL-8 in regulating melanoma growth and metastasis. So, IL-8 could be targeted to arrest melanoma growth and metastasis in-vivo. Hence, IL-8 could be a potential target for melanoma treatment.

Up-Regulation of Activating Transcription Factor 3 in Human Fibroblasts Inhibits Melanoma Cell Growth and Migration Through a Paracrine Pathway.

The treatment of melanoma has remained a difficult challenge. Targeting the tumor stroma has recently attracted attention for developing novel strategies for melanoma therapy. Activating transcription factor 3 (ATF3) plays a crucial role in regulating tumorigenesis and development, but whether the expression of ATF3 in human dermal fibroblasts (HDFs) can affect melanoma development hasn't been studied. Our results show that ATF3 expression is downregulated in stromal cells of human melanoma. HDFs expressing high levels of ATF3 suppressed the growth and migration of melanoma cells in association with downregulation of different cytokines including IL-6 in vitro. In vivo, HDFs with high ATF3 expression reduced tumor formation. Adding recombinant IL-6 to melanoma cells reversed those in vitro and in vivo effects, suggesting that ATF3 expression by HDFs regulates melanoma progression through the IL-6/STAT3 pathway. More importantly, HDFs pretreated with cyclosporine A or phenformin to induce ATF3 expression inhibited melanoma cell growth in vitro and in vivo. In summary, our study reveals that ATF3 suppresses human melanoma growth and that inducing the expression of ATF3 in HDFs can inhibit melanoma growth, a new potential melanoma therapeutic approach.

Tacrolimus and ascomycin inhibit melanoma cell growth, migration and invasion via targeting nuclear factor of activated T-cell 3.

Melanoma is the most malignant form of skin cancer with high metastatic potential. Nuclear factor of activated T-cells (NFATs) are discovered as transcription factors that regulate the expression of proinflammatory cytokines and other genes during the immune response. Among five NFAT members, NFAT3 is exclusively not expressed in immune cells and its role in progression of different types of cancer remains controversial. Our previous study showed that NFAT3 was highly expressed in skin cancer compared with normal skin tissues and critical for melanoma cell survival and tumor growth. Here, we reported that knockdown of NFAT3 expression, as well as treatment with the calcineurin (CaN) inhibitors, tacrolimus (FK506) or ascomycin (FK520) inhibits melanoma cell migration and invasion, and also proliferation and colony formation. Mechanistic studies revealed that FK506 or FK520 blocked the nuclear translocation and reduced the transcriptional activity of NFAT3. These data support that the antimelanoma effect of FK506 and FK520 is partially mediated by inhibiting the oncogenic factor NFAT3, suggesting that therapeutics based on NFAT3 inhibition may be effective in clinical melanoma treatment.

Can Interleukin‐8 Alone be Responsible for Regulating Melanoma Growth & Metastasis? In‐vitro studies based on treatments of human melanoma cell lines (BLM and 1205Lu) with sex steroids, IL‐8, curcumin and active vitamin‐D3

Previous clinical studies indicated that menstruating females were better protected in melanoma than post‐menopausal women and men of any age. But, the mechanism responsible for protection in menstruating females was not investigated. In addition, epidemiological data showed an increased male mortality in melanoma. Our in‐vitro study showed female sex hormone progesterone significantly inhibited human melanoma cell growth. Elisarray showed that progesterone action was mediated by a specific suppression of pro‐inflammatory cytokine IL‐8. So, the effect of male sex hormones androstenedione (AD) and testosterone (T) on melanoma cell growth was checked. AD and T also suppressed cell growth and IL‐8 secretion. But, addition of progesterone (10μM) along with androgens showed an additive effect on the inhibition of melanoma cell growth and IL‐8 suppression. This observation gave the idea of protective function of progesterone in melanoma, which was experimentally observed by a significant decrease in in‐vitro migration and adhesion functions. As steroids (P, AD, T) targeted IL‐8 for their actions, effect of vit‐D3 on melanoma cells was checked. Active form of vitamin‐D3 (25 μM) also targeted IL‐8 and decreased cell growth, suggesting IL‐8 could be regulating melanoma cell growth. In order to check whether IL‐8 regulated melanoma cell growth, IL‐8 was directly added to melanoma cells and endogenous IL‐8 was suppressed by 2 hrs pre‐treatment with curcumin. Addition of IL‐8 (1 ng/ml) to 1205Lu melanoma cells stimulated cell growth to 117%, whereas suppression of IL‐8 by curcumin (100 μM) pre‐treatment decreased cell growth to 24%. Similar results were obtained with BLM cells. This brought IL‐8 into focus as an important molecule regulating melanoma cell growth. Further in‐vitro experiments to restore curcumin (25 μM) pre‐treated melanoma cell growth (71.5%) by the addition of IL‐8 (100 ng/ml) resulted in a near complete restoration of cell growth (95.3%) in 1205Lu melanoma cells, suggesting IL‐8 could be the molecule regulating melanoma cell growth. Similar results were observed with BLM cells. In addition, in‐vivo conditional expression of IL‐8 in nude mouse by Dr. Singh et al., indicated involvement of IL‐8 in melanoma growth and metastasis.ConclusionBoth in‐vitro and in‐vivo studies indicated that IL‐8 alone could be responsible for regulating melanoma growth and metastasis. Hence, IL‐8 could be targeted to arrest melanoma growth and metastasis in‐vivo.

In Vitro Biophysical and Biological Characterization of Lipid Nanoparticles Co-Encapsulating Oncosuppressors miR-199b-5p and miR-204-5p as Potentiators of Target Therapy in Metastatic Melanoma.

Uncontrolled MAPK signaling is the main oncogenic driver in metastatic melanomas bearing mutations in BRAF kinase. These tumors are currently treated with the combination of BRAF/MEK inhibitors (MAPKi), but this therapy is plagued by drug resistance. In this context we recently discovered that several microRNAs are involved in the development of drug resistance. In particular miR-204-5p and miR-199b-5p were found to function as antagonists of resistance because their enforced overexpression is able to inhibit melanoma cell growth in vitro either alone or in combination with MAPKi. However, the use of miRNAs in therapy is hampered by their rapid degradation in serum and biological fluids, as well as by the poor intracellular uptake. Here, we developed lipid nanoparticles (LNPs) encapsulating miR-204-5p, miR-199b-5p individually or in combination. We obtained LNPs with mean diameters < 200 nm and high miRNA encapsulation efficiency. These formulations were tested in vitro on several melanoma cell lines sensitive to MAPKi or rendered drug resistant. Our results show that LNPs encapsulating combinations of the two oncosuppressor miRNAs are highly efficient in impairing melanoma cell proliferation and viability, affect key signaling pathways involved in melanoma cell survival, and potentiate the efficacy of drugs inhibiting BRAF and MEK. These results warrant further assessment of the anti-tumor efficacy of oncosuppressor miRNAs encapsulating LNPs in in vivo tumor models.

Procyanidin C1 Inhibits Melanoma Cell Growth by Activating 67-kDa Laminin Receptor Signaling.

Procyanidin C1 (PC1) is an epicatechin trimer found mainly in grapes that is reported to provide several health benefits. However, little is known about the molecular mechanisms underlying these benefits. The aim of this study is to demonstrate the molecular mechanisms by which PC1 operates. A 67-kDa laminin receptor (67LR) is identified as a cell surface receptor of PC1, with a Kd value of 2.8µm. PC1 induces an inhibitory effect on growth, accompanied by dephosphorylation of the C-kinase potentiated protein phosphatase-1 inhibitor protein of 17kDa (CPI17) and myosin regulatory light chain (MRLC) proteins, followed by actin cytoskeleton remodeling in melanoma cells. These actions are mediated by protein kinase A (PKA) and protein phosphatase 2A (PP2A) activation once PC1 is bound to 67LR. It is demonstrated that PC1 elicits melanoma cell growth inhibition by activating the 67LR/PKA/PP2A/CPI17/MRLC pathway.

A novel chalcone derivative has antitumor activity in melanoma by inducing DNA damage through the upregulation of ROS products

BackgroundMelanoma is one of the most aggressive tumors with the remarkable characteristic of resistance to traditional chemotherapy and radiotherapy. Although targeted therapy and immunotherapy benefit advanced melanoma patient treatment, BRAFi (BRAF inhibitor) resistance and the lower response rates or severe side effects of immunotherapy have been observed, therefore, it is necessary to develop novel inhibitors for melanoma treatment.MethodsWe detected the cell proliferation of lj-1-59 in different melanoma cells by CCK 8 and colony formation assay. To further explore the mechanisms of lj-1-59 in melanoma, we performed RNA sequencing to discover the pathway of differential gene enrichment. Western blot and Q-RT-PCR were confirmed to study the function of lj-1-59 in melanoma.ResultsWe found that lj-1-59 inhibits melanoma cell proliferation in vitro and in vivo, induces cell cycle arrest at the G2/M phase and promotes apoptosis in melanoma cell lines. Furthermore, RNA-Seq was performed to study alterations in gene expression profiles after treatment with lj-1-59 in melanoma cells, revealing that this compound regulates various pathways, such as DNA replication, P53, apoptosis and the cell cycle. Additionally, we validated the effect of lj-1-59 on key gene expression alterations by Q-RT-PCR. Our findings showed that lj-1-59 significantly increases ROS (reactive oxygen species) products, leading to DNA toxicity in melanoma cell lines. Moreover, lj-1-59 increases ROS levels in BRAFi -resistant melanoma cells, leading to DNA damage, which caused G2/M phase arrest and apoptosis.ConclusionsTaken together, we found that lj-1-59 treatment inhibits melanoma cell growth by inducing apoptosis and DNA damage through increased ROS levels, suggesting that this compound is a potential therapeutic drug for melanoma treatment.

Ubiquitination of P53 by E3 ligase MKRN2 promotes melanoma cell proliferation.

Melanoma is the most aggressive and lethal type of skin cancer. The aim of the present study was to illustrate the molecular mechanism of makorin ring finger protein 2 (MKRN2) control of melanoma cell proliferation. The expression level of MKRN2 was detected in human malignant melanoma cell lines by immunoblotting and reverse transcription-quantitative PCR. Short hairpin RNAs for MKRN2 were designed and transfected into melanoma cells, and the proliferation of these cells was detected by MTT and colony formation assays. The interaction of MKRN2 with P53 was detected by co-immunoprecipitation and glutathione S-transferase pulldown assays. The ubiquitination of P53 by MKRN2 was detected by in vitro ubiquitination assays. A P53-knockout cell line was generated using the CRISPR-Cas9 method. MKRN2 exhibited higher expression levels in melanoma cells, and downregulation of MKRN2 inhibited melanoma cell growth in a P53-dependent manner. MKRN2 regulated melanoma cell proliferation by interacting and ubiquitylating P53, which suggests that MKRN2 may be a potential therapeutic target for melanoma.

Dibutyltin(IV) and Tributyltin(IV) Derivatives of meso-Tetra(4-sulfonatophenyl)porphine Inhibit the Growth and the Migration of Human Melanoma Cells.

Melanoma is the most aggressive and deadly form of skin cancer, which is largely due to its propensity to metastasize. Therefore, with the aim to inhibit the growth and the metastatic dissemination of melanoma cells and to provide a novel treatment option, we studied the effects of the melanoma treatment with two organotin(IV) complexes of the meso-tetra(4-sulfonato-phenyl)porphine, namely (Bu2Sn)2TPPS and (Bu3Sn)4TPPS. In particular, we showed that nanomolar concentrations of (Bu2Sn)2TPPS and (Bu3Sn)4TPPS are sufficient to inhibit melanoma cell growth, to increase the expression of the full-length poly (ADP-ribose) polymerase (PARP-1), to induce the cell cycle arrest respectively at G2/M and G0/G1 through the inhibition of the Cyclin D1 expression and to inhibit cell colony formation. Nanomolar concentrations of (Bu2Sn)2TPPS and (Bu3Sn)4TPPS are also sufficient to inhibit the melanoma cell migration and the expression of some adhesion receptors. Moreover, we report that (Bu2Sn)2TPPS and (Bu3Sn)4TPPS act downstream of BRAF, mainly bypassing its functions, but targeting the STAT3 signalling protein. Finally, these results suggest that (Bu2Sn)2TPPS and (Bu3Sn)4TPPS may be effective therapeutic strategies for their role in the inhibition of melanoma growth and migration.

Benserazide is a novel inhibitor targeting PKM2 for melanoma treatment.

The M2 splice isoform of pyruvate kinase (PKM2) is a key enzyme for generating pyruvate and ATP in the glycolytic pathway, whereas the role of PKM2 in tumorigenesis remains a subject of debate. In our study, we found PKM2 is highly expressed in melanoma patients and the malignance is positively correlated with high PKM2 activity and glycolytic capability in melanoma cells. Suppression of PKM2 expression by knocking down markedly attenuated malignant phenotype both in vitro and in vivo, and restoration of PKM2 expression in PKM2 depleted cells could rescue melanoma cells proliferation, invasion and metastasis. With the data indicating PKM2 as a potential therapeutic target, we performed screening for PKM2 inhibitors and identified benserazide (Ben), a drug currently in clinical use. We demonstrated that Ben directly binds to and blocks PKM2 enzyme activity, leading to inhibition of aerobic glycolysis concurrent up-regulation of OXPHOS. Of note, despite PKM2 is very similar to PKM1, Ben does not affect PKM1 enzyme activity. We showed that Ben significantly inhibits cell proliferation, colony formation, invasion and migration in vitro and in vivo. The specificity of Ben was demonstrated by the findings that, suppression of PKM2 expression diminishes the efficacy of Ben in inhibition of melanoma cell growth; ectopic PKM2 expression in normal cells sensitizes cells to Ben treatment. Interestingly, PKM2 activity and aerobic glycolysis are upregulated in BRAFi-resistant melanoma cells. As a result, BRAFi-resistant cells exhibit heightened sensitivity to suppression of PKM2 expression or treatment with Ben both in vitro and in vivo.

Cyanidin-3-o-Glucoside Pharmacologically Inhibits Tumorigenesis via Estrogen Receptor β in Melanoma Mice.

Expression patterns of estrogen receptors [ERα, ERβ, and G-protein associated ER (GPER)] in melanoma and skin may suggest their differential roles in carcinogenesis. Phytoestrogenic compound cyanidin-3-o-glucoside (C3G) has been shown to inhibit the growth and metastatic potential of melanoma, although the underlying molecular mechanism remains unclear. The aim of this study was to clarify the mechanism of action of C3G in melanoma in vitro and in vivo, as well as to characterize the functional expressions of ERs in melanoma. In normal skin or melanoma (n = 20/each), no ERα protein was detectable, whereas expression of ERβ was high in skin but weak focal or negative in melanoma; and finally high expression of GPER in all skin vs. 50% melanoma tissues (10/20) was found. These results correspond with our analysis of the melanoma survival rates (SRs) from Human Protein Atlas and The Cancer Genome Atlas GDC (362 patients), where low ERβ expression in melanoma correlate with a poor relapse-free survival, and no correlations were observed between SRs and ERα or GPER expression in melanoma. Furthermore, we demonstrated that C3G treatment arrested the cell cycle at the G2/M phase by targeting cyclin B1 (CCNB1) and promoted apoptosis via ERβ in both mouse and human melanoma cell lines, and inhibited melanoma cell growth in vivo. Our study suggested that C3G elicits an agonistic effect toward ERβ signaling enhancement, which may serve as a potential novel therapeutic and preventive approach for melanoma.

4'-Bromo-resveratrol, a dual Sirtuin-1 and Sirtuin-3 inhibitor, inhibits melanoma cell growth through mitochondrial metabolic reprogramming.

Sirtuin-1 and -3 (SIRT1 and SIRT3) are important nicotinamide adenine dinucleotide (NAD+ )-dependent deacetylases known to regulate a variety of cellular functions. Studies have shown that SIRT1 and SIRT3 were overexpressed in human melanoma cells and tissues and their inhibition resulted in a significant antiproliferative response in human melanoma cells and antitumor response in a mouse xenograft model of melanoma. In this study, we determined the antiproliferative efficacy of a newly identified dual small molecule inhibitor of SIRT1 and SIRT3, 4'-bromo-resveratrol (4'-BR), in human melanoma cell lines (G361, SK-MEL-28, and SK-MEL-2). Our data demonstrate that 4'-BR treatment of melanoma cells resulted in (a) decrease in proliferation and clonogenic survival; (b) induction of apoptosis accompanied by a decrease in procaspase-3, procaspase-8, and increase in the cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP); (c) marked downregulation of proliferating cell nuclear antigen (PCNA); and (d) inhibition of melanoma cell migration. Further, 4'-BR caused a G0/G1 phase arrest of melanoma cells that was accompanied by an increase in WAF-1/P21 and decrease in Cyclin D1/Cyclin-dependent kinase 6 protein levels. Furthermore, we found that 4'-BR causes a decrease in lactate production, glucose uptake, and NAD+ /NADH ratio. These responses were accompanied by downregulation in lactate dehydrogenase A and glucose transporter 1 in melanoma cells. Collectively, our data suggest that dual inhibition of SIRT1 and SIRT3 using 4'-BR imparted antiproliferative effects in melanoma cells through a metabolic reprogramming and affecting the cell cycle and apoptosis signaling. Therefore, concomitant pharmacological inhibition of SIRT1 and SIRT3 needs further investigation for melanoma management.

Abstract 4289: ERK3 negatively regulates IL-6/STAT3 signaling via SOCS3 in cancer cells

Abstract Extracellular signal-regulated kinase 3 (ERK3) is an atypical mitogen activated protein kinase (MAPK) which is dysregulated in several cancers including lung cancer, breast cancer and melanoma. It has a single Ser-Glu-Gly (SEG) phospho-acceptor motif in its activation loop, instead of the Thr-Xaa-Tyr (TXY) motif conserved in the classical MAPKs. Moreover, compared to the conventional MAPKs, much less is known about the targets of ERK3 signaling. Hence, to elucidate the ERK3-regulated genes and pathways, we performed gene microarray analyses of both A549 lung cancer cells and A375 melanoma cells after treatment with either a siRNA specifically targeting ERK3 or a non-targeting control siRNA. Interestingly, IL-6 target genes were found to be highly upregulated upon ERK3 knockdown. Interleukin-6 (IL-6) is a pleiotropic cytokine which signals via the JAK/STAT3 pathway. Phosphorylation of STAT3 at Tyrosine 705 (Y705) by JAK activates STAT3 in response to IL-6 stimulation. IL-6 signaling is negatively regulated by a feedback inhibitor SOCS3 (Suppressor of cytokine signaling 3), which binds to and inhibits JAK kinase activity. Interestingly, we noted that ERK3 interacts with SOCS3 from a published Yeast-two-hybrid screening. We validated the interaction between ERK3 and SOCS3 by co-immunoprecipitation assay. In addition, we found that ERK3 downregulates the phosphorylation of STAT3 at Y705. In line with its negative regulation of oncogenic STAT3 activity, ERK3 inhibits melanoma cell growth, migration/invasion and colony formation in soft agar. Taken together, we have revealed a novel role for ERK3 in suppressing IL-6/STAT3 signaling via SOCS3 in cancer cells. Citation Format: Astha Shakya, Minyi Chen, Michael Markey, Weiwen Long. ERK3 negatively regulates IL-6/STAT3 signaling via SOCS3 in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4289.

Effect of Sulforaphane and 5-Aza-2'-Deoxycytidine on Melanoma Cell Growth.

Background: UV exposure-induced oxidative stress is implicated as a driving mechanism for melanoma. Increased oxidative stress results in DNA damage and epigenetic dysregulation. Accordingly, we explored whether a low dose of the antioxidant sulforaphane (SFN) in combination with the epigenetic drug 5-aza-2’-deoxycytidine (DAC) could slow melanoma cell growth. SFN is a natural bioactivated product of the cruciferous family, while DAC is a DNA methyltransferase inhibitor. Methods: Melanoma cell growth characteristics, gene transcription profiles, and histone epigenetic modifications were measured after single and combination treatments with SFN and DAC. Results: We detected melanoma cell growth inhibition and specific changes in gene expression profiles upon combinational treatments with SFN and DAC, while no significant alterations in histone epigenetic modifications were observed. Dysregulated gene transcription of a key immunoregulator cytokine—C-C motif ligand 5 (CCL-5)—was validated. Conclusions: These results indicate a potential combinatorial effect of a dietary antioxidant and an FDA-approved epigenetic drug in controlling melanoma cell growth.

MiR‑21 inhibitor facilitates the anticancer activity of doxorubicin loaded nanometer in melanoma.

MicroRNA‑21 (miR‑21) is a potential therapeutic target for melanoma. Whether miR‑21 inhibitor affects the anti‑cancer activity of doxorubicin assisted by c(RGDyK)‑modified liposome (DLN) in melanoma and the underlying mechanisms are largely unknown. In this study, in vitro and animal models were used to explore the effect of DLN combined with miR‑21 inhibitor on melanoma cells. The data demonstrated that treatment with 5 µl DLN (final concentration of doxorubicin 5 mg/ml) for 72 h effectively inhibited melanoma cell growth (~75% inhibition). The experiments were then divided into five groups: Control group, vector group, DLN group, miR‑21 inhibitor group and miR‑21 inhibitor + DLN group. Compared with the control group, DLN (5 µl) or miR‑21 inhibitor significantly reduced migration and invasion of melanoma cells, promoted apoptosis and arrested cells at the G1 phase. Notably, the combined application of DLN with miR‑21 inhibitor further promoted the anti‑cancer effects (reducing migration and invasion of melanoma cells, promoting apoptosis and arresting cells at G1 phase) compared with individual application of DLN or miR‑21 inhibitor. Mechanically, DLN did not function by reducing miR‑21 expression, whereas DLN and miR‑21 inhibitor downregulated B‑cell lymphoma-2 (BCL‑2) expression, and facilitated BCL‑2‑associated X protein (Bax) and P53 expression in melanoma cells. DLN and miR‑21 inhibitor together displayed stronger effects on Bcl‑2, Bax and P53 expression that each alone. In vivo data further demonstrated that DLN inhibited tumor growth further than a similar dose of doxorubicin only. Furthermore, miR‑21 inhibitor and DLN exerted the optimal anti‑cancer effect compared with single application of DLN or miR‑21 inhibitor. Together, the findings demonstrated miR‑21 inhibitor facilitated the anti‑cancer activity of DLN in melanoma, and the mechanisms involved Bcl‑2, Bax and P53 expression.