The Anticancer Potential of Doxycycline and Minocycline—A Comparative Study on Amelanotic Melanoma Cell Lines

Malignant melanoma is still a serious medical problem. Relatively high mortality, a still-growing number of newly diagnosed cases, and insufficiently effective methods of therapy necessitate melanoma research. Tetracyclines are compounds with pleiotropic pharmacological properties. Previously published studies on melanotic melanoma cells ascertained that minocycline and doxycycline exerted an anti-melanoma effect. The purpose of the study was to assess the anti-melanoma potential and mechanisms of action of minocycline and doxycycline using A375 and C32 human amelanotic melanoma cell lines. The obtained results indicate that the tested drugs inhibited proliferation, decreased cell viability, and induced apoptosis in amelanotic melanoma cells. The treatment caused changes in the cell cycle profile and decreased the intracellular level of reduced thiols and mitochondrial membrane potential. The exposure of A375 and C32 cells to minocycline and doxycycline triggered the release of cytochrome c and activated initiator and effector caspases. The anti-melanoma effect of analyzed drugs appeared to be related to the up-regulation of ERK1/2 and MITF. Moreover, it was noticed that minocycline and doxycycline increased the level of LC3A/B, an autophagy marker, in A375 cells. In summary, the study showed the pleiotropic anti-cancer action of minocycline and doxycycline against amelanotic melanoma cells. Considering all results, it could be concluded that doxycycline was a more potent drug than minocycline.

Nuclear Localization of BRAFV600E Is Associated with HMOX-1 Upregulation and Aggressive Behavior of Melanoma Cells

Background: Previously, we have demonstrated that nuclear BRAFV600E is associated with melanoma aggressiveness and vemurafenib resistance. However, the underlying mechanisms of how nuclear localization of BRAFV600E promotes cell aggressiveness have not yet been investigated. Despite therapeutic advancements targeting cutaneous melanoma, unknown cellular processes prevent effective treatment for this malignancy, prompting an urgent need to identify new biological targets. This study aims to explore the association of inducible heme oxygenase 1 (HMOX-1) with nuclear BRAFV600E in promoting melanoma aggressiveness. Methods: Proteomics analysis was performed to identify the interacting partner(s) of nuclear BRAFV600E. Immunohistochemistry was applied to evaluate the levels of HMOX-1 and nuclear BRAFV600E expression in melanoma and adjacent healthy tissues. Immunofluorescence assessed the nuclear localization of BRAFV600E in vemurafenib-resistant A375R melanoma cells. Further study of HMOX-1 knockdown or BRAFV600E overexpression in melanoma cells suggested a role for HMOX-1 in the regulation of cell proliferation in vivo and in vitro. Finally, Western blot analysis was performed to confirm the pathway by which HMOX-1 mediates Akt signaling. Results: Proteomics results showed that HMOX-1 protein expression was 10-fold higher in resistant A375R cells compared to parental counterpart cells. In vitro and in vivo results illustrate that nuclear BRAFV600E promotes HMOX-1 overexpression, whereas HMOX-1 reduction represses melanoma cell proliferation and tumor growth. Mechanistic studies revealed that HMOX-1 was associated with nuclear BRAFV600E localization, thus promoting melanoma proliferation via a persistent activation of the AKT pathway. Conclusions: Our results highlight a previously unknown mechanism in which the nuclear BRAFV600E/HMOX-1/AKT axis plays an essential role in melanoma cell proliferation. Targeting HMOX-1 could be a novel method for treating melanoma patients who develop BRAF inhibitor resistance.

MicroRNA-603 Promotes Progression of Cutaneous Melanoma by Regulating TBX5

Background. Although studies manifested that microRNA-603 plays a vital role in many cancers, the modulatory mechanism of microRNA-603 in cutaneous melanoma remains unknown. We aimed to investigate the roles of microRNA-603 in cutaneous melanoma cells. Methods. First, microRNA-603 expression in cutaneous melanoma cell lines was detected by qRT-PCR. The mRNA and protein expression levels of TBX5 in cutaneous melanoma cell lines were tested by qRT-PCR and western blot, respectively. In addition, the interaction between microRNA-603 and TBX5 was determined by dual-luciferase reporter gene assay, and their impacts on the growth of cutaneous melanoma cells were detected by cellular function experiments such as MTT, colony formation, and Transwell assays. Results. The expression level of microRNA-603 in human cutaneous melanoma cells was relatively upregulated. Overexpressing microRNA-603 could promote progression of cutaneous melanoma cells, while silencing microRNA-603 expression could suppress the malignant progression of cutaneous melanoma. In addition, TBX5 was lowly expressed in cutaneous melanoma cells. As confirmed by dual-luciferase assay, microRNA-603 could specifically bind to 3 ′ UTR of TBX5 and regulate TBX5. The results of the rescue experiment demonstrated that inhibiting microRNA-603 expression could suppress the proliferation, migration, and invasion of cutaneous melanoma cells, but its suppressive effect could be restored by TBX5. Conclusion. MicroRNA-603 could regulate the expression of TBX5, thus promoting the malignant progression of cutaneous melanoma cells.

A Novel Small-Molecule Inhibitor Displays Its Therapeutic Potency Through Mitochondria-Mediated Apoptosis And Autophagy In Melanoma

Melanoma is the most aggressive skin cancer with high mortality. It is vital to develop novel low toxicity drugs with anti-proliferation activity and metastasis suppressive activity in melanoma. Here, we reported a novel anti-tumor drug SCZ0148, and then investigated its inhibition effect on melanoma. The anticancer efficacy of SCZ0148 was confirmed by using cytotoxicity test, colony formation assay, wound-healing assay, cell apoptosis detection, mitochondrial potential assay, reactive oxygen species (ROS) production and western-blot analysis. The cytotoxicity test showed that SCZ0148 inhibited melanoma cell lines proliferation in a dose- and time-dependent manner without obvious toxicity and side effects on normal cells. The results of the colony formation assay were in agreement with the cytotoxicity test. In addition, SCZ0148 induced melanoma cell apoptosis and promoted cell destructive autophagy through the ROS-mediated mitochondrial apoptosis pathway. Notably, SCZ0148 significantly inhibited the migration of melanoma cells through the matrix metalloprotein 9 (MMP-9) mediated pathway. In conclusion, these findings suggest that SCZ0148 may be a potential therapeutic drug to inhibit the proliferation and metastasis of melanoma.

Vitamin D Enhances Anticancer Properties of Cediranib, a VEGFR Inhibitor, by Modulation of VEGFR2 Expression in Melanoma Cells

Regardless of the recent groundbreaking introduction of personalized therapy, melanoma continues to be one of the most lethal skin malignancies. Still, a substantial proportion of patients either fail to respond to the therapy or will relapse over time, representing a challenging clinical problem. Recently, we have shown that vitamin D enhances the effectiveness of classical chemotherapeutics in the human malignant melanoma A375 cell line. In search for new combination strategies and adjuvant settings to improve melanoma patient outcomes in the current study, the effects of cediranib (AZD2171), an oral tyrosine kinase inhibitor of VEGFR1-3, PDGFR, and c-KIT, used in combination either with 1,25(OH)2D3 or with low-calcemic analog calcipotriol were tested on four human malignant melanoma cell lines (A375, MNT-1, RPMI-7951, and SK-MEL-28). Melanoma cells were pretreated with vitamin D and subsequently exposed to cediranib. We observed a marked decrease in melanoma cell proliferation (A375 and SK-MEL-28), G2/M cell cycle arrest, and a significant decrease in melanoma cell mobility in experimental conditions used (A375). Surprisingly, concurrently with a very desirable decrease in melanoma cell proliferation and mobility, we noticed the upregulation of VEGFR2 at both protein and mRNA levels. No effect of vitamin D was observed in MNT-1 and RPMI-7951 melanoma cells. It seems that vitamin D derivatives enhance cediranib efficacy by modulation of VEGFR2 expression in melanoma cells expressing VEGFR2. In conclusion, our experiments demonstrated that vitamin D derivatives hold promise as novel adjuvant candidates to conquer melanoma, especially in patients suffering from vitamin D deficiency. However, further extensive research is indispensable to reliably assess their potential benefits for melanoma patients.

Predicting the possible effect of miR-203a-3p and miR-29a-3p on DNMT3B and GAS7 genes expression

Aberrant expression of genes involved in methylation, including DNA methyltransferase 3 Beta (DNMT3B), can cause hypermethylation of various tumor suppressor genes. In this regard, various molecular factors such as microRNAs can play a critical role in regulating these methyltransferase enzymes and eventually downstream genes such as growth arrest specific 7 (GAS7). Accordingly, in the present study we aimed to predict regulatory effect of miRNAs on DNMT3B and GAS7 genes expression in melanoma cell line. hsa-miR-203a-3p and hsa-miR-29a-3p were predicted and selected using bioinformatics software. The Real-time PCR technique was performed to investigate the regulatory effect of these molecules on the DNMT3B and GAS7 genes expression. Expression analysis of DNMT3B gene in A375 cell line showed that there was a significant increase compared to control (p value = 0.0015). Analysis of hsa-miR-203a-3p and hsa-miR-29a-3p indicated the insignificant decreased expression in melanoma cell line compared to control (p value < 0.05). Compared to control, the expression of GAS7 gene in melanoma cells showed a significant decrease (p value = 0.0323). Finally, our findings showed that the decreased expression of hsa-miR-203a-3p and hsa-miR-29a-3p can hypothesize that their aberrant expression caused DNMT3B dysfunction, possible methylation of the GAS7 gene, and ultimately decreased its expression. However, complementary studies are necessary to definite comment.

Study on the Anti-cancer Potential of Aegle marmelos Fruit Extract Pro and Anti Apoptotic Molecule in Human Melanoma Cell Line-A375

Aegle marmelos is also known as bael which is commonly found in south East Asia and Indian-sub continent. The origin of bael is India. Bael is also known as the golden apple, Bengal-quince in India. In the ancient medical system, Aegle marmelos play an important role and its extract is also useful in inflammation, diabetes, cancer and asthma. The leaves are used for anti-inflammatory, nervous disorder, control blood sugar and fruit is used to treat antiviral, anti-diabetics, and brain and heart tonic. In addition, studies have proved that bael is used for the treatment and prevention of cancer. The main aim of this study is to assess the anti-cancer potential of Aegle marmelos fruit extract pro and anti apoptotic molecules in human melanoma cell line-A375. In the present study, Human Melanoma A375 cells will be produced, grown and will be passed in different culture flasks, then RNA isolation was done and by reverse transcriptase process, RNA gets converted into cDNA. This cDNA will be used for the amplification of growth factor beta using gene specific primers by commercially available real time PCR kit. The anticancer potential effect was found in 400µg/ml, as the concentration increases, the cell viability is decreased. The current study explains the potential application of bael in pharmacological and medicinal uses in near future.