Human Melanoma Cell Lines Research Articles

Singlet-oxygen photosensitizers with a tetrad structure and a single BODIPY chromophore: An evidence for transition state stabilization of intersystem crossing

Eleven tetrad Boron dipyrromethene (BODIPY) derivatives were synthesized and examined as singlet-oxygen photosensitizers (PSs). It was clearly shown that a charge shifted state (CSS) with a twisted π-bond makes a considerable contributed to the transition state (TS) stabilization at the minimum energy seam of crossing (MESX) for intersystem crossing (ISC). An EC50 of less than 10 nM was achieved for the G361 human melanoma cell line using one of the investigated PSs, which were only composed of 1st− and 2nd-period elements with compact structures.

484 Melatonin induces melanoma cell death and synergizes with BRAF/MEK inhibitors by altering cell bioenergetics

Melanoma is a leading cause of cancer deaths worldwide. Although targeted therapy and immunotherapy have improved the outcome of patients with metastatic disease, unwanted side effects are a problem. Herein, by using human melanoma cell lines in vitro, we explore that melatonin enhances anti-tumor activity of commonly used BRAF/MEK inhibitors, i.e. vemurafenib (VF) and cobimetinib (CB), respectively. Our results have demonstrated that compared with VF/CB alone, melatonin significantly reduced proliferation read-outs (colony assay, drop assay) and induced of apoptosis (cleaved Casp-3, PARP) in melanoma cells.

HSPB8 counteracts tumor activity of BRAF- and NRAS-mutant melanoma cells by modulation of RAS-prenylation and autophagy

Cutaneous melanoma is one of the most aggressive and lethal forms of skin cancer. Some specific driver mutations have been described in multiple oncogenes including BRAF and NRAS that are mutated in 60–70% and 15–20% of melanoma, respectively. The aim of this study was to evaluate the role of Small Heat Shock Protein B8 (HSPB8) on cell growth and migration of both BLM (BRAFwt/NRASQ61R) and A375 (BRAFV600E/NRASwt) human melanoma cell lines. HSPB8 is a member of the HSPB family of chaperones involved in protein quality control (PQC) system and contributes to chaperone assisted selective autophagy (CASA) as well as in the regulation of mitotic spindle. In cancer, HSPB8 has anti- or pro-tumoral action depending on tumor type. In melanoma cell lines characterized by low HSPB8 levels, we demonstrated that the restoration of HSPB8 expression causes cell growth arrest, reversion of EMT (Epithelial-Mesenchymal Transition)-like phenotype switching and antimigratory effect, independently from the cell mutational status. We demonstrated that HSPB8 regulates the levels of the active prenylated form of NRAS in NRAS-mutant and NRAS-wild-type melanoma cell lines. Consequently, the inhibition of NRAS impairs the activation of Akt/mTOR pathway inducing autophagy activation. Autophagy can play a dual role in regulating cell death and survival. We have therefore demonstrated that HSPB8-induced autophagy is a crucial event that counteracts cell growth in melanoma. Collectively, our results suggest that HSPB8 has an antitumoral action in melanoma cells characterized by BRAF and NRAS mutations.

Comparison of the Anticancer Effects of Arvanil and Olvanil When Combined with Cisplatin and Mitoxantrone in Various Melanoma Cell Lines-An Isobolographic Analysis.

Due to the unique structures of arvanil and olvanil, the drugs combine certain properties of both cannabinoids and vanilloids, which makes them able to stimulate both TPRV1 and CB1 receptors and causes them to be interesting agents in the setting of carcinoma treatment. The aim of this study was to investigate the cytotoxic and anti-proliferative effects of arvanil and olvanil when administered alone and in combination with cisplatin (CDDP) and mitoxantrone (MTX), using various primary (A375, FM55P) and metastatic (SK-MEL 28, FM55M2) human malignant melanoma cell lines. The results indicate that both arvanil and olvanil inhibited (dose-dependently) the viability and proliferation of various malignant melanoma cells, as demonstrated by MTT and BrdU assays. The safety profile of both arvanil and olvanil tested in human keratinocytes (HaCaT) and normal human melanocytes (HEMa-LP) revealed that neither arvanil nor olvanil caused significant cytotoxicity in HaCaT and HEMa-LP cell lines in LDH and MTT assays. Isobolographically, it was found that both arvanil and olvanil exerted additive interactions with MTX and antagonistic interactions with CDDP in the studied malignant melanoma cell lines. In conclusion, the combinations of arvanil or olvanil with MTX may be considered as a part of melanoma multi-drug therapy; however, the combination of these compounds with CDDP should be carefully considered due to the antagonistic interactions observed in the studied malignant melanoma cell lines.

Pharmaco-Toxicological Assessment of the Combined Cytotoxic Effects of Digoxin and Betulinic Acid in Melanoma Cells.

Betulinic acid, a small molecule from pentacyclic triterpenes class, has been widely studied for its antitumor activity, revealing that it induces the apoptosis of tumor cells in a selective manner. In recent years, digoxin, a cardiac glycoside found particularly in the plant species Digitalis lanata, has drawn interest for its potential antitumor properties. The present study was designed to evaluate the antimelanoma potential of betulinic acid (BA), digoxin (DG), and their association (DG + BA). In vitro assessments were performed 24 h post-treatment on two human melanoma cell lines (SK-Mel-28 and RPMI-7951). In addition, the potential irritant effects of the test samples were evaluated using the chorioallantoic membrane of hen's eggs. BA and DG exhibit a concentration-dependent cytotoxic activity, with the combination of the two having a more marked effect on the decrease in cell viability (~17% for SK-Mel-28 cells and ~23% for RPMI-7951 cells). Further, morphological changes (rounding of the cells and their separation from the plaque) and alterations in the nucleus and actin fibers (condensation of chromatin and actin fibers, formation of apoptotic bodies) were observed, indicating an apoptotic-like process. Moreover, no irritating effects were observed in ovo. As a result, DG + BA acid may have synergistic potential in the antitumor treatment of melanoma, but future studies are needed in order to clarify the biological mechanisms involved.

Effects of Piptoporus betulinus Ethanolic Extract on the Proliferation and Viability of Melanoma Cells and Models of Their Cell Membranes.

Piptoporus betulinus is a fungus known for its medicinal properties. It possesses antimicrobial, anti-inflammatory, and anti-cancer activity. In this study, several tests were performed to evaluate the cytotoxic effect of the ethanolic extract of Piptoporus betulinus on two melanoma human cell lines, WM115 primary and A375 metastatic cell lines, as well as Hs27 human skin fibroblasts. The extract proved to affect cancer cells in a dose-dependent manner, and at the same time showed a low cytotoxicity towards the normal cells. The total phenolic content (TPC) was determined spectrophotometrically by the Folin-Ciocalteu method (F-C), and the potential antioxidant activity was measured by ferric-reducing antioxidant power (FRAP) assay. One of the active compounds in the extract is betulin. It was isolated and then its cytotoxic activity was compared to the results obtained from the Piptoporus betulinus extract. To further understand the mechanism of action of the extract's anticancer activity, tests on model cell membranes were conducted. A model membrane of a melanoma cell was designed and consisted of 1,2-dimyristoyl-sn-glycero-3-phosphocholine, disialoganglioside-GD1a and cholesterol: DMPC:GD1a:chol (5:2:3 mole ratio). Changes in a Langmuir monolayer were observed and described based on Π-Amol isotherm and compressibility modulus changes. LB lipid bilayers were deposited on a hydrophilic gold substrate and analyzed by IR and X-ray photoelectron spectroscopy. Our study provides new data on the effect of Piptoporus betulinus extract on melanoma cells and its impact on the model of melanoma plasma membranes.

Orthopalladated N,N-dimethyl-1-phenethylamine compounds containing 2,6-lutidine: Synthesis, DNA binding studies and cytotoxicity evaluation

Metathetical reactions involving [Pd(C2,N-dmpa)(μ-Cl)]2 (1) and the appropriate halides/pseudohalides salts afforded cyclopalladated dimers of the type [Pd(C2,N-dmpa)(μ-X)]2 (dmpa = S(−) enantiomer of N,N-dimethyl-1-phenethylamine; {X = Cl (1) and N3 (2)}. Mononuclear compounds of general formulae [Pd(C2,N-dmpa)(X)(lut)] {X = Cl (1a) and N3 (2a)} were obtained by bridge-splitting reactions involving the corresponding [Pd(C2,N-dmpa)(μ-X)]2 with 2,6-lutidine (lut) in the 1:2 M ratio at room temperature. Both the cyclopalladated compounds were characterized by means of elemental analysis, FT-IR, Raman, 1H and 13C NMR spectroscopy. The antiproliferative activity of the mononuclear compounds 1a-2a was evaluated towards human glioblastoma (U251 and T98G) and melanoma cell lines (HT144 and LB373) and their IC50 values determined between 1 and 6 μM. In most cases, the cytotoxic effects of compounds 1a-2a showed to be similar to those of cisplatin (depending on the cell line), what is of utmost importance when considering treatment alternatives for these aggressive tumor types. Binding studies on the representative compound 2a towards ct-DNA, however, showed low or no affinity, suggesting that the observed cytotoxicity against the human cell lines may involve different mechanisms of action compared to that of the platinum-based chemotherapy drug. The ability of 1a to induce the inhibition of topoisomerase IIα activity has also been investigated. These cyclopalladated compounds can be transported and distributed through the body by human serum albumin (HSA), as observed by competition and computational studies with the protein. These findings are very promising and have motivated further studies for the design of new and bioactive cyclopalladated compounds for future medicinal purposes.

Integrative Transcriptomic Analysis Identify Potential m6A Pathway-Related Drugs That Inhibit Cancer Cell Proliferation

Recent studies have found that m6A modification of mRNA may play important roles in the progression of various types of cancers. However, current knowledge about drugs that can interfere with m6A methylation and inhibit cancer cell proliferation is still far from comprehensive. To this end, we performed integrative analysis on transcriptome data with perturbation of m6A writers or erasers and identified consensus m6A-related differentially expressed genes (DEGs). Comparative analysis of these m6A-related DEGs with Connectivity Map signatures highlight potential m6A-targeted drugs. Among them, we experimentally verified the inhibitory effects of AZ628 on the proliferation of human breast cancer cell lines and R428 on the proliferation of human melanoma cell lines. Both drugs can significantly reduce the cellular level of m6A modification. These results suggest an m6A-related new target pathway by AZ628 and R428 and provide new candidate m6A-related drugs that inhibit cancer cell proliferation.

ODP555 In-vitro Combo Treatments of Human Melanoma (1205Lu, WM238) Cell Lines with Curcumin, Vitamins and Steroids

Abstract Previous combo treatment experiments on BLM human melanoma cell line identified two combos vit-A + RU-486 and Cur+P+RU as the best combinations to decrease melanoma cell growth as well as IL-8 secretion in -vitro. In order to confirm this finding, experiments were repeated on two other human melanoma cell lines 1205Lu and WM238. Initial single or solo compound treatment on 1205Lu cells resulted in cell growth in the range of 37–59% compared to untreated control 1205Lu cell growth at 100%. A double combo treatment with combinations such as D+P, D+RU, A+P, A+RU, A+D resulted in cell growth in the range of 17 to 37% on the 1205Lu cell line. Whereas on WM238 cell line resulted in cell growth in between 18–22%. A triple combo such as combinations of three compounds (Cur+P+RU, Cur+A+P, Cur+D+RU) resulted in cell growth in the range of 19-30% on 1205Lu cells, whereas on WM238 cells resulted in between 19–20% cell growth. Elisarray of the supernatants from the combos which showed a significant decrease in cell growth revealed the regulation only in IL-8 secretion. IL-8 secretion in the supernatants of combos treated with A+RU and Cur+P+RU was17.5 and 8. 0 pg/ml respectively on the 1205Lu cell line. Whereas, IL-8 secretion was 0.515 and 0.926 pg/ml respectively in the supernatants of A+RU and Cur+P+RU combo treatments on the WM238 cell line. Conclusion Based on cell growth, Elisarray and IL-8 quantitation, two combos A+RU and Cur+P+RU also fared well on the two other cell lines 1205Lu and WM238. Presentation: No date and time listed

Cytotoxic profile study, DNA and protein binding activity of a new dinuclear nickel(II) thiocyanato complex

One new binuclear Ni(II) complex with the molecular formula [Ni2(L)2(SCN)2(μ-SCN)2] (1) has been synthesized using a tridentate Schiff base ligand (L), formed by the condensation between quinoline-2-carboxaldehyde and N,N-dimethylethylenediamine. Complex 1 was characterized by FT-IR, UV-vis, and ESI-mass spectroscopy techniques, as well as elemental analysis, cyclic voltammetry, and single-crystal X-ray crystallography. The X-ray diffraction analysis of 1 indicated a centrosymmetric complex with nickel(II) centers in a distorted octahedral geometry and double bridged by thiocyanate anions via end-to-end (μ-1,3) fashion. The interaction of 1 with CT-DNA was explored by UV-vis and fluorescence spectroscopy, viscosity and cyclic voltammetry which indicated a moderate intercalation of the complex with CT-DNA. The protein binding study of 1 with BSA and HSA was investigated by various spectroscopic methods and indicated a static quenching process. In addition, 1 showed prominent in vitro cytotoxic activity against human melanoma (A375) and human breast carcinoma (MDA-MB-231) cell lines under the identical condition with IC50 value of 149.06 and 124.22 µM, respectively.

Response of Skin-Derived and Metastatic Human Malignant Melanoma Cell Lines to Thymoquinone and Thymoquinone-Loaded Liposomes.

Thymoquinone has been proved to be effective against neoplasms, including skin cancer. Its high lipophilicity, however, may limit its potential use as a drug. Melanoma remains the deadliest of all skin cancers worldwide, due to its high heterogeneity, depending on the stage of the disease. Our goal was to compare the anti-cancer activity of free thymoquinone and thymoquinone-loaded liposomes on two melanoma cell lines that originated from different stages of this cancer: skin-derived A375 and metastatic WM9. We evaluated the proapoptotic effects of free thymoquinone by flow cytometry and Western blot, and its mitotoxicity by means of JC-1 assay. Additionally, we compared the cytotoxicity of free thymoquinone and thymoquinone in liposomes by WST-1 assay. Our results revealed a higher antiproliferative effect of TQ in WM9 cells, whereas its higher proapoptotic activity was observed in the A375 cell line. Moreover, the thymoquinone-loaded liposome was proved to exert stronger cytotoxic effect on both cell lines studied than free thymoquinone. Differences in the response of melanoma cells derived from different stages of the disease to thymoquinone, as well as their different responses to free and carrier-delivered thymoquinone, are essential for the development of new anti-melanoma therapies. However, further research is required to fully understand them.

Establishment and Characterisation of Human Clear Cell Sarcoma and Malignant Melanoma Cell Lines.

Clear cell sarcoma of soft tissue (CCSST) and conventional malignant melanoma (MM) are rare and aggressive tumours with similarities in morphology and the expression of melanocytic markers. We established two CCSST cell lines (FU-CCSST-1 and FU-CCSST-2) from soft tissues of the patella and supraclavicular. A MM cell line (FU-MM-1) was established from lymph node metastases of subungual malignant melanoma. FU-CCSST-2 cells were transplantable to immunodeficient mice. Immunohistochemical studies demonstrated tumour cells were negative for cytokeratin AE1/AE3 and positive for S100 protein, HMB45, Melan-A, CD146 and SOX10 in all specimens. FU-CCSST-1 and FU-CCSST-2 harboured t(12;22)(q13;q12) translocations with expression of the EWSR1/ATF1 fusion gene. FU-MM-1 demonstrated loss of the short arm of chromosome 9 and harboured wild-type BRAF (codon 469 and 600) and NRAS (codon 12, 13 and 61). We report the establishment and characterisation of CCSST and MM cell lines that may have utility in the study of pathogenic mechanisms and development of novel therapeutic reagents.

Tyrosinase inhibition of major secondary metabolites isolated from endangered Meconopsis bhutanica Tosh.Yoshida & Grey-Wilson

Meconopsis bhutanica Tosh.Yoshida & Grey-Wilson ( Meconopsis Vig., fam: Papaveraceae), is a Critically Endangered species utilized in Tibetan medicine. Due to the very limited numbers of wild individuals, the chemical and biological basis of its medicinal value has not been studied. The objective of this study was to explore the chemical constituents of Meconopsis bhutanica and to determine the antityrosinase activity and influence on human melanoma cell lines A375. The chemical constituents were mainly isolated through column chromatography and semipreparative high performance liquid chromatography , and the structures were elucidated by nuclear magnetic resonance spectra. The inhibition on tyrosinase was examined by microplate reader. High performance liquid chromatography, spectrofluorophotometer, and molecular docking were used to analyze the effects on the function and structure of tyrosinase. The influence on A375 cells were measured by the Cell Counting Kit. Finally, a total of eleven phenolic compounds were isolated and identified from Meconopsis bhutanica . All of these compounds were found in this plant for the first time. The results of antityrosinase activity assay showed that compound 9 exhibited tyrosinase inhibition obviously. Furthermore, compound 9 could decrease the oxidation products and fluorescence emission intensity of tyrosinase. However, compound 9 had no effect on the proliferation of human melanoma A375 cells at high concentrations. The present paper uncovered the chemical composition of Meconopsis bhutanica and assessed the tyrosinase inhibition activity, which will provide data for comparing bioactive chemical constituents between the wild and cultivated ones. This study paved the way for the conservation, cultivation, and application of Meconopsis bhutanica . • The tyrosinase inhibition constituents of Meconopsis bhutanica were first studied. • (E)-2-(β- D -glucopyranosyloxy)-4-methoxycinnamic acid exhibited tyrosinase inhibition. • Meconopsis bhutanica had application value as regulators of tyrosinase activity. • The distribution of endangered Meconopsis bhutanica in China was first discovered. • Meconopsis bhutanica demonstrated potential to be cultivated.

Cytotoxic Neoflavonoids and Chalcones from the Heartwood of Dalbergia melanoxylon

Ten compounds, consisting of neoflavonoids (1-5), isoflavonoids (6 and 7), flavanone (8), and chalcones (9 and 10) were isolated from the ethyl acetate and n-butanol-soluble fractions of the heartwood of Dalbergia melanoxylon. The chemical structures were identified on the basis of spectroscopic evidence and compared to previously reported spectra. Compounds 1-10 were evaluated for cytotoxicity against HCT116 human colorectal cancer, MDA-MB-231 human metastatic breast cancer, and A2058 human melanoma cell lines. Among them, compounds 3 and 10 showed the strongest cytotoxic activity with IC50 values of 11.92 ± 1.07 μM, 10.83 ± 1.02 μM, and 14.37 ± 1.02 μM, 13.62 ± 1.09 μM against HCT116 and MDA-MB-231 cell lines, respectively. Compounds 9 and 10 also had cytotoxic activity with IC50 values of 13.49 ± 1.18 μM and 9.82 ± 0.91 μM against A2058 cell lines, respectively. To the best our knowledge, compounds 2 and 5-10 were isolated from this source for the first time.

Guanine inhibits the growth of human glioma and melanoma cell lines by interacting with GPR23.

Guanine-based purines (GBPs) exert numerous biological effects at the central nervous system through putative membrane receptors, the existence of which is still elusive. To shed light on this question, we screened orphan and poorly characterized G protein-coupled receptors (GPRs), selecting those that showed a high purinoreceptor similarity and were expressed in glioma cells, where GBPs exerted a powerful antiproliferative effect. Of the GPRs chosen, only the silencing of GPR23, also known as lysophosphatidic acid (LPA) 4 receptor, counteracted GBP-induced growth inhibition in U87 cells. Guanine (GUA) was the most potent compound behind the GPR23-mediated effect, acting as the endpoint effector of GBP antiproliferative effects. Accordingly, cells stably expressing GPR23 showed increased sensitivity to GUA. Furthermore, while GPR23 expression was low in a hypoxanthine-guanine phosphoribosyl-transferase (HGPRT)-mutated melanoma cell line showing poor sensitivity to GBPs, and in HGPRT-silenced glioma cells, GPR23-induced expression in both cell types rescued GUA-mediated cell growth inhibition. Finally, binding experiments using [3H]-GUA and U87 cell membranes revealed the existence of a selective GUA binding (KD = 29.44 ± 4.07 nM; Bmax 1.007 ± 0.035 pmol/mg prot) likely to GPR23. Overall, these data suggest GPR23 involvement in modulating responses to GUA in tumor cell lines, although further research needs to verify whether this receptor mediates other GUA effects.

WZB117 enhanced the anti-tumor effect of apatinib against melanoma via blocking STAT3/PKM2 axis.

Background: Melanoma is the most lethal skin malignant tumor with a short survival once stepping into the metastatic status and poses a therapeutic challenge. Apatinib (a tyrosine kinase inhibitor) is a promising antiangiogenic agent for the treatment of metastatic melanoma. However, antiangiogenic monotherapy is prone to acquired drug resistance and has a limited therapeutic effect. The persistence dependence of glycolytic metabolism in antiangiogenic therapy-resistant cells provides evidence that glycolysis inhibitors may enhance the effect of antiangiogenic therapy. So, this study aimed to investigate whether WZB117 (a specific GLUT1 inhibitor) could enhance the anti-tumor effect of apatinib against melanoma and its potential mechanisms. Methods: We investigated the anti-tumor effects of apatinib alone or in combination with WZB117 on human melanoma cell lines (A375 and SK-MEL-28). The MTT assay determined cell viability and the half-maximal inhibitory concentration (IC50). Multiple drug effect/combination indexes (CI) analysis was conducted to assess interactions between apatinib and WZB117. Signal transducer and activator of transcription 3 (STAT3) pathway measured by western blotting and immunofluorescence staining. RNA expression analyses were performed using the reverse transcription-quantitative PCR method. Results: Apatinib and WZB117 showed dose and time-dependent growth inhibitory effects in both melanoma cells. The IC50 of apatinib at 48 h in A375 and SK-MEL-28 cells was 62.58 and 59.61 μM, respectively, while the IC50 of WZB117 was 116.85 and 113.91 μM, respectively. The CI values of the two drugs were 0.538 and 0.544, respectively, indicating a synergistic effect of apatinib combined with WZB117. We also found that glucose consumption and lactate production were suppressed by apatinib plus WZB117 in a dose-dependent manner, paralleled by reducing glycolytic enzyme pyruvate kinase M2 (PKM2). The potential mechanism of the combination was to suppress the phosphorylation of STAT3. Knockdown of STAT3 by siRNA inhibited the expression of PKM2, while the activation of STAT3 by IL-6 increased the expression of PKM2. The effects of IL-6 were attenuated by apatinib combined with WZB117 treatment. Conclusion: WZB117 enhanced the anti-tumor effect of apatinib against melanoma via modulating glycolysis by blocking the STAT3/PKM2 axis, which suggested the combination of apatinib with WZB117 could be a potential therapeutic candidate for melanoma.

Raptinal Induces Gasdermin E-Dependent Pyroptosis in Naïve and Therapy-Resistant Melanoma.

Raptinal can rapidly induce pyroptosis in naïve and BRAFi plus MEKi-resistant melanoma, which may be beneficial for patients who have developed acquired resistance to targeted therapies.

Topical systems for the controlled release of antineoplastic Drugs: Oxidized Alginate-Gelatin Hydrogel/Unilamellar vesicles

The efficacy of chemotherapeutic procedures relies on delivering proper concentrations of anti-cancer drugs in the tumor surroundings, so as to prevent potential side effects on healthy tissues. Novel drug carrier platforms should not just be able to deliver anticancer molecules, but also allow for adjustements in the way these drugs are administered to the patients. We developed a system for delivering water-insoluble drugs, based on the use of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), or bis(2-ethylhexyl) sulfosuccinate benzyl-n-hexadecyldimethylammonium (BHD-AOT), embedded into oxidized alginate-gelatin (ADA/Gel) hydrogel, emulating a patch for topic applications. After being loaded with curcumin, cancer cells such as human colorectal adenocarcinoma (HCT116 and DLD-1) and melanoma cell lines (MEL501), and non-malignant cells such as mammary epithelial cell lines (NMuMG) and embryonal fibroblasts (NIH 3T3 or NEO cells) were analyzed for biocompatibility and cytotoxic effects. The results show that the proposed system can load comparatively higher concentrations of the drug (with respect to other nano/microcarriers in the literature), and that it can enhance the likelihood of the drug being uptaken by cancer cells instead of non-malignant cells. These assays were complemented by diffusion studies across the stratum corneum of rat skin, with the aim of determining the system’s efficiency during topical application. Finally, the stability of the patch was tested after lyophilization to determine its potential pharmaceutical use. As a whole, the combined system represents a highly reliable and robust method for embedding and delivering complex insoluble chemotherapeutical molecules, and it is less invasive than other alternative methods in the literature.

Imiquimod-induced ROS production causes lysosomal membrane permeabilization and activates caspase-8-mediated apoptosis in skin cancer cells

BackgroundLysosomal cell death is induced by lysosomal membrane permeabilization (LMP) and the subsequent release of lysosomal proteolytic enzymes, including cathepsins (CTSs), which results in mitochondrial dysfunction and apoptosis. Imiquimod (IMQ), a synthetic TLR7 ligand, has both antiviral and antitumor activity against various skin malignancies in clinical treatment. Previously, we demonstrated IMQ not only caused lysosomal dysfunction but also triggered lysosome biogenesis to achieve lysosomal adaptation in cancer cells. ObjectiveTo determine whether lysosomes are involved in IMQ-induced apoptosis. MethodsThe human skin cancer cell lines BCC, A375 and mouse melanoma cell line B16F10 were used in all experiments. Cell death was determined by the Cell Counting Kit-8 (CCK-8) assay and DNA content assay. Protein expression was determined by immunoblotting. Caspase-8 activity was assessed using a fluorescence caspase-8 kit and determined by flow cytometry and confocal microscopy. ResultsIMQ not only induced lysosome damage but also abrogated lysosome function in skin cancer cells. IMQ-induced caspase-8 activation contributed to the processes of lysosomal cell death. Moreover, the use of ROS scavengers significantly abolished caspase-8 activation and inhibited IMQ-induced LMP. Additionally, pharmacological inhibition of CTSD not only abrogated caspase-8 activation but also rescued IMQ-induced cell death. Finally, lysosome-alkalizing agents enhanced the cytotoxicity of IMQ in vitro and in vivo. ConclusionsIMQ-induced ROS accumulation promotes LMP, releases CTSs into the cytosol, stimulates caspase-8 activation and finally causes lysosomal cell death. Lysosomal cell death and the CTSD/caspase-8 axis may play a crucial role in IMQ-induced cell death.

Additive Interactions between Betulinic Acid and Two Taxanes in In Vitro Tests against Four Human Malignant Melanoma Cell Lines.

The incidence of melanoma is steadily increasing worldwide. Melanoma is the most lethal skin cancer, and new therapeutic methods are being sought. Our research aimed to investigate the cytotoxic and antiproliferative effects of betulinic acid in vitro, used alone and in combination with taxanes (paclitaxel, docetaxel) in four melanoma cell lines. Isobolographic analysis allowed us to assess the interactions between these compounds. Betulinic acid had no cytotoxic effect on normal human keratinocyte HaCaT cells; the amount of LDH released by them was significantly lower compared to melanoma cell lines. The present study shows that betulinic acid significantly inhibits the growth of melanoma cell lines in vitro. The IC50 values of betulinic acid ranged from 2.21 µM to 15.94 µM against the four melanoma lines. Co-treatment of betulinic acid with paclitaxel or docetaxel generated desirable drug–drug interactions, such as an additive and additive with a tendency to synergy interactions.