Apoptosis In B16 Melanoma Cells Research Articles

Pueraria protein extract inhibits melanogenesis and promotes melanoma cell apoptosis through the regulation of MITF and mitochondrial‑related pathways.

PuerariaLobata Radix (P.Lobata Radix) is an edible traditional Chinese medicine that contains various active compounds. Proteins from P.Lobata Radix have become the subject of increased interest in recent years. In evaluating the whitening effect on the skin, the present study found that the P.Lobata Radix water‑soluble total protein extract (PLP) had the strongest inhibitory effect on tyrosinase activity. In the present study, the anti‑melanogenic effect of PLP and the inhibitory effect on B16 melanoma cells were investigated. PLP significantly reduced the tyrosinase activity and melanin content in B16 melanoma cells. Mechanistically, PLP inhibited melanogenesis by decreasing the expression of tyrosinase, tyrosinase‑related protein (TRP)‑1 and TRP‑2 through downregulation of the microphthalmia‑associated transcription factor (MITF) gene, which was mediated by inhibition of p38 mitogen‑activated protein kinase signaling. In addition, PLP inhibited cell viability and triggered apoptosis of B16 cells in a dose‑dependent manner. Exposure to PLP reduced the mitochondrial membrane potential (MMP) and decreased ATP generation, leading to mitochondria‑related apoptosis of B16 melanoma cells. The expression levels of succinate dehydrogenase (SDH) and its two related subunits (SDHA and SDHB) were downregulated significantly by PLP, which may be associated with the regulation of mitochondrial energy metabolism by PLP. These results may explain why MMP collapse and reduced ATP generation were observed in B16 melanoma cells treated with PLP. Finally, the present study demonstrated that the inhibition of melanin synthesis by PLP was correlated with the regulation of antioxidant enzymes to reduce reactive oxygen species levels. These results suggested that PLP inhibits melanogenesis by downregulating the expression of MITF‑related melanogenic enzymes and triggering apoptosis through mitochondria‑related pathways.

Conjugate of ibrutinib with a TLR7 agonist suppresses melanoma progression and enhances antitumor immunity.

The use of large molecules for immunotherapy has led to exciting developments in cancer treatment, such as the development of PD-1/PD-L1 antibodies. However, small molecule targeted therapies still lack effective immune-functional classes. Ideal anticancer drugs should simultaneously generate immune memory when killing cancer cells to prevent tumor relapse and metastasis. To this end, we carried out a rationally designed strategy to develop novel classes of small molecule compounds with bifunctional targeting and immunostimulatory abilities by conjugating targeting compounds with TLR7 agonists, generating immune-targeting conjugates (ImmunTacs). GY161, as a representative ImmunTac, was synthesized via chemical conjugation of ibrutinib with a TLR7 agonist. In vitro, GY161 stimulated the production of cytokines by mouse spleen lymphocytes, promoted the maturation of dendritic cells (DCs), and inhibited the growth and induced the apoptosis of B16 melanoma cells by regulating the c-Met/β-catenin pathway. In vivo, GY161 enhanced the frequency of CD8+ T cells in spleens and tumors, suppressed the growth of B16 melanoma cell-derived tumors and prolonged the survival time of mice. In summary, GY161 could prevent melanoma progression through direct tumor killing and by triggering specific immunity. These results strongly suggest that ImmunTacs are a reliable and promising strategy for developing small molecule immunogenic anticancer drugs.

Bromodomain-containing protein 4 inhibitor JQ1 promotes melanoma cell apoptosis by regulating mitochondrial dynamics.

Although the role of bromodomain‐containing protein 4 (BRD4) in ovarian cancer, pancreatic cancer, lymphoma, and many other diseases is well known, its function in cutaneous melanoma is only partially understood. The results of the present study show that the BRD4 inhibitor JQ1 promotes the apoptosis of B16 melanoma cells by altering mitochondrial dynamics, thereby inducing mitochondrial dysfunction and increasing oxidative stress. We found that treatment of B16 cells with different concentrations of JQ1 (125 nmol/L or 250 nmol/L) significantly downregulated the expression of protein subunits involved in mitochondrial respiratory chain complexes I, III, IV, and V, increased reactive oxygen species, induced energy metabolism dysfunction, significantly enhanced apoptosis, and activated the mitochondrial apoptosis pathway. At the same time, JQ1 inhibited the activation of AMP‐activated protein kinase, a metabolic energy sensor. In addition, we found that the mRNA and protein levels of mitochondrial dynamin‐related protein 1 increased, whereas the levels of mitochondrial fusion protein 1 and optic atrophy protein 1 decreased. Mechanistically, we determined that JQ1 inhibited the expression of c‐Myc and altered mitochondrial dynamics, eventually leading to changes in the mitochondrial function, metabolism, and apoptosis of B16 melanoma cells.

Hydroxyurea regulates the development and survival of B16 Melanoma Cells by upregulating MiR-7013-3p.

miRNAs are a family of short, noncoding RNAs that are involved in many processes in melanoma cells. MITF acts as a master regulator of melanocyte function, development and survival by modulating various genes. Hydroxyurea (HU) is used to treat melanoma, and miRNA expression is altered after HU treatment in B16 melanoma cells. In this study, we screened for miRNAs that were upregulated after HU treatment and that targeted the MITF gene. We found that miR-7013-3p exhibited increased expression after HU treatment and could bind to MITF. miR-7013-3p inhibited melanin production, proliferation, and migration and promoted apoptosis in B16 melanoma cells. The results may provide more information on the roles of miR-7013-3p in B16 melanoma cells.

Oxygen partial pressure plays a crucial role in B16 melanoma cell survival by regulating autophagy and mitochondrial functions

The oxygen partial pressure generally increases when cancerous cells become part of the blood vessels. The study was to investigate the influence of oxygen partial pressure on the apoptosis of B16 melanoma cells. Our results demonstrated that both short-term and long-term hypoxia/reoxygenation (H/R) treatment increased stress-induced intracellular reactive oxygen species (ROS). H/R treatment also increased apoptosis and autophagy in B16 cells. N-acetylcysteine (NAC), a ROS scavenger, can reduce ROS and aid survival. However, Bafilomycin A1, an autophagy inhibitor, can accelerate cell death. Thus, our work revealed that ROS and autophagy play critical roles in cellular H/R.

Characterization and Inducing Melanoma Cell Apoptosis Activity of Mannosylerythritol Lipids-A Produced from Pseudozyma aphidis.

Mannosylerythritol lipids (MELs) are natural glycolipid biosurfactants which have potential applications in the fields of food, cosmetic and medicine. In this study, MELs were produced from vegetable oil by Pseudozyma aphidis. Their structural data through LC/MS, GC/MS and NMR analysis revealed that MEL-A with two acetyls was the major compound and the identified homologs of MEL-A contained a length of C8 to C14 fatty acid chains. This glycolipid exhibited a surface tension of 27.69 mN/m at a critical micelle concentration (CMC), self-assembling into particles in the water solution. It was observed to induce cell growth-inhibition and apoptosis of B16 melanoma cells in a dose-dependent manner, as well as cause cell cycle arrest at the S phase. Further quantitative RT-PCR analysis and western blotting revealed an increasing tendency of both mRNA and protein expressions of Caspase-12, CHOP, GRP78 and Caspase-3, and a down-regulation of protein Bcl-2. Combined with the up regulation of signaling IRE1 and ATF6, it can be speculated that MEL-A-induced B16 melanoma cell apoptosis was associated with the endoplasmic reticulum stress (ERS).

Dietary sulforaphane inhibits histone deacetylase activity in B16 melanoma cells

Dietary sulforaphane (SF) exerts anticancer effects via epigenetic changes resulting from the inhibition of histone deacetylase (HDAC) activity in colon and prostate cancer cells. The present study aimed to evaluate the inhibition of HDAC activity by SF and its effect on apoptosis in B16 melanoma cells. SF significantly inhibited proliferation of B16 melanoma cells with an IC50 of 6.1 ± 1.0 µM, as assessed with the MTT assay performed after SF treatment for 48 h. SF induced apoptosis in B16 cells in a dose-dependent manner and caused them to undergo G2/M arrest. By quantitative real-time PCR and western blot analysis, expression of p21 and Bax was upregulated in B16 cells treated for 48 h with SF, and the levels of acetylated histones H3 and H4 were increased. In conclusion, these findings suggest that SF as functional food ingredients may be effective against melanoma through inhibition of HDAC activity.

Characterization and assessment of nanoencapsulated sanguinarine chloride as a potential treatment for melanoma

Sanguinarine has a history of use in both folk medicine and early dermatology for the treatment of cutaneous neoplasms. Applied indiscriminately, bloodroot is an escharotic agent with potential to cause extensive tissue necrosis. However, when used in a controlled fashion, sanguinarine imparts selective cytotoxic/anti-proliferative activity through multiple mechanisms against human/ murine melanoma. To exploit sanguinarine's observed activity against melanoma, a targeted delivery system is required. We present a sol-gel based nanoparticulate platform for encapsulating sanguinarine chloride(sang-np)-a targeted therapeutic capable of steady, reliable delivery of predictable quantities of drug over a sustained time period with minimal undesirable effects. Size and release kinetics of sang-np were characterized using dynamic light scattering and ultraviolet-visible spectroscopy respectively. In vitro efficacy of sang-np was assessed. At both 2 and 24 hours, free sanguinarine killed > 90% of B16 melanoma cells, assessed via MTT assay. At 2 hours, sang-np killed a portion of melanoma cells, increasing to percentages comparable to free sanguinarine by 24 hours. Control(empty) nanoparticles exerted minimal toxicity to melanoma cells at both time points. TUNEL assay revealed that treatment with both sanguinarine and sang-np induces apoptosis in B16 melanoma cells, suggesting that both treatments act via the same mechanism of action. These data confirm controlled release of sanguinarine from sang-np, as well as comparable efficacy and mechanism of action to sanguinarine alone. This suggests that nanoparticle delivery of sanguinarine may be a unique approach to capitalize on this potent agent's inherent anti-tumor activity and overcome many of the limitations with its current formulation.

Proteomic study reveals a co-occurrence of gallic acid-induced apoptosis and glycolysis in B16F10 melanoma cells.

Gallic acid (GA) has long been associated with a wide range of biological activities. In this study, its antitumor effect against B16F10 melanoma cells was demonstrated by MTT assay, cell migration assay, wound-healing assay, and flow cytometric analysis. GA with a concentration >200 μM shows apoptotic activity toward B16F10 cells. According to Western blotting data, overexpressions of cleaved forms of caspase-9, caspase-3, and PARP-1 and pro-apoptotic Bax and Bad, accompanied by underexpressed anti-apoptotic Bcl-2 and Bcl-xL indicate that GA induces B16F10 cell apoptosis via mitochondrial pathway. The 2-DE based comparative proteomics was further employed in B16F10 cells with and without GA treatment for a large-scale protein expression profiling. A total of 41 differential protein spots were quantified, and their identities were characterized using LC-MS/MS analysis and database matching. In addition to some regulated proteins that were associated with apoptosis, interestingly, some identified proteins involved in glycolysis such as glucokinase, α-enolase, aldolase, pyruvate kinase, and GAPDH were simultaneously up-regulated, which reveals that the GA-induced cellular apoptosis in B16 melanoma cells is associated with metabolic glycolysis.

Atractylenolide II induces G1 cell-cycle arrest and apoptosis in B16 melanoma cells

Atractylenolide II (AT-II) is a sesquiterpene compound isolated from the dried rhizome of Atractylodes macrocephala (Baizhu in Chinese), which is traditionally prescribed for melanoma treatment by Chinese medicine practitioners. Our previous study showed that AT-II can inhibit B16 cells proliferation. Here we investigate the mechanistic basis for the anti-proliferative activity of AT-II in B16 melanoma cells. Cell viability was examined by MTT assay. Cell cycle distribution and apoptosis were determined by flow cytometry. Protein expression was determined by Western blotting. AT-II treatment for 48 h dose-dependently inhibited cell proliferation with an IC(50) of 82.3 μM, and induced G1 phase cell cycle arrest. Moreover, treatment with 75 μM AT-II induced apoptosis. These observations were associated with the decrease of the expression of Cdk2, phosphorylated-Akt, phosphorylated-ERK and Bcl-2, the increase of the expression of phosphorylated-p38, phosphorylated-p53, p21, p27, and activation of caspases-8, -9 and -3. In addition, a chemical inhibitor of p53, PFTα, significantly decreased AT-II-mediated growth inhibition and apoptosis. We demonstrated that the G1-arresting and apoptotic effects of AT-II in B16 cells involve p38 activation as well as ERK and Akt inactivation, and the cytotoxic/apoptotic effects of AT-II are potentially p53 dependent. These findings provided chemical and pharmacological basis for the traditional application of Baizhu in melanoma treatment.

Polyamine transporter recognization and antitumor effects of anthracenymethyl homospermidine

This study was conducted to examine the polyamine transporter (PAT) recognization and antitumor effects of anthracenymethyl homospermidine (ANTMHspd) and its apoptotic mechanism in B16 melanoma cells. ANTMHspd promoted a dose-dependent apoptosis in B16 melanoma cells and the apoptosis was associated with the excellent PAT recognization, externalization of cell membrane phosphatidylserine and the disruption of mitochondria, these processes were correlated with up-regulation of polyamine oxidase, an increase in intracellular reactive oxygen species (ROS) production and oxidative stress. In addition, reduction of MMP, release of cytochrome c, up-regulation of Bax protein expression, down-regulation of Bcl-2 protein expression, and activation of caspase-3, caspase-9 were also observed in B16 cells after treatment with ANTMHspd. Furthermore, N-acetylcysteine obviously antagonized ANTMHspd-induced apoptosis. Importantly, ANTMHspd was found to be better tolerated and revealed potent antitumor effect on inhibiting tumor growth in situ and suppressing pulmonary metastasis in xenograft mice model. These data demonstrate that ANTMHspd is an excellent PAT recognization and potent antitumor agent.

Anticancer effect of realgar nanoparticles on mouse melanoma skin cancer in vivo via transdermal drug delivery

Realgar has been used successfully to treat diseases for thousands of years, but its poor water solubility and high toxicity hampered its further medical uses. Here, we first applied transdermal drug delivery system to deliver realgar nanoparticles to investigate its anticancer effect and toxicity in vivo. In this study, MTT assay and flow cytometry analysis demonstrated that realgar significantly suppressed the proliferation and induced apoptosis of B16 melanoma cells in a dose-dependent manner. Transdermal penetration studies in vitro showed realgar nanoparticles could be delivered efficiently through skin. Tests on tumor-bearing C57BL/6 mice displayed that realgar could decrease the tumor volume markedly via transdermal drug delivery compared with the intraperitoneal administration and the control. Hematoxylin-eosin and immunohistochemical staining revealed that it could inhibit angiogenesis. The monitoring of the hepatic injury, body weight, feeding behavior, motor activity, and skin irritation of each animal indicated little toxicity of realgar to mice. The results demonstrated that realgar nanoparticles can be dermally delivered to achieve high efficacy against menaloma in vivo with low toxicity.

Sphingosine 1-Phosphate Triggers Apoptotic Signal for B16 Melanoma Cells via ERK and Caspase Activation

The bioactive sphingolipid metabolite sphingosine 1-phosphate (S1P), recently was reported to induce apoptosis of some cancer cells and neurons, although it generally known to exert mitogenic and antiapoptotic effects. In this study, we investigated the effects of S1P on the cell growth, melanogenesis, and apoptosis of cultured B16 mouse melanoma cells. In results, S1P was found to induce apoptosis in B16 melanoma cells in a dose- and time-dependent manner, but exerted minimal effects on melanogenesis. Although receptors of sphingosine 1-phosphate (endothelial differentiation gene 1 [Edg]/S1P1, Edg5/S1P2, Edg3/S1P3) were expressed in B16 melanoma cells, they were shown not to be associated with S1P-induced apoptosis. In addition, pertussis toxin did not block the apoptotic effects of S1P on B16 melanoma cells. S1P induced caspase-3 activation and the extracellular signal-regulated kinase (ERK) activation. Interestingly, the ERK pathway inhibitor, UO126, reversed the apoptotic effects of S1P on B16 melanoma cells. These results suggest that S1P induced apoptosis of B16 melanoma cells via an Edg receptor-independent, pertussis toxin-insensitive pathway, and appears to be associated with the ERK and caspase-3 activation.

Sodium Ascorbate induces apoptosis in neuroblastoma cell lines by interfering with iron uptake

BackgroundNeuroblastoma (NB) is an extra-cranial solid tumour of childhood. In spite of the good clinical response to first-line therapy, complete eradication of NB cells is rarely achieved. Thus, new therapeutic strategies are needed to eradicate surviving NB cells and prevent relapse. Sodium ascorbate has been recently reported to induce apoptosis of B16 melanoma cells through down-regulation of the transferrin receptor, CD71. Since NB and melanoma share the same embryologic neuroectodermal origin, we used different human NB cell lines to assess whether the same findings occurred.ResultsWe could observe dose- and time-dependent induction of apoptosis in all NB cell lines. Sodium ascorbate decreased the expression of CD71 and caused cell death within 24 h. An increase in the global and specific caspase activity took place, as well as an early loss of the mitochondrial transmembrane potential. Moreover, intracellular iron was significantly decreased after exposure to sodium ascorbate. Apoptotic markers were reverted when the cells were pretreated with the iron donor ferric ammonium citrate (FAC), further confirming that iron depletion is responsible for the ascorbate-induced cell death in NB cells.ConclusionSodium ascorbate is highly toxic to neuroblastoma cell lines and the specific mechanism of vitamin C-induced apoptosis is due to a perturbation of intracellular iron levels ensuing TfR-downregulation.

Phenothiazines induce apoptosis in a B16 mouse melanoma cell line and attenuate in vivo melanoma tumor growth

Phenothiazines and related antipsychotics were reported to have an antiproliferative effect in several tissue cultures. The aims of this study were: a) to screen in vitro, the potential anti-cancer activity of phenothiazines in wild-type and multi-drug resistant (MDR) B16 mouse melanoma cell lines; and b) to determine the in vivo anti-tumor effect of an in vitro selected highly potent phenothiazine (thioridazine) in a murine melanoma model. The following phenothiazines were evaluated: perphenazine, fluphenazine, thioridazine trifluoperazine and chlorpromazine. All agents induced a dose-dependent decrease in cell viability in wild-type and in MDR B16 melanoma cells. Thioridazine displayed the highest antiproliferative activity. Flow cytometric analyses of 24-h treated B16 melanoma cells revealed an increase in fragmented DNA (16.3 vs 71.3% and 87.2% in controls, 25 microM and 50 microM thioridazine-treated, respectively). Apoptosis was confirmed by co-staining of thioridazine-treated B16 cells (12.5 microM) with propidium iodide and Hoechst 33342 reagents. Caspase-3 expression, a typical mediator of apoptosis, was markedly increased following a 4-h exposure of B16 cells to thioridazine (25 microM and 50 microM). This increase could be blocked by a specific caspase-3 inhibitor. In vivo studies were performed using female C57/Bl mice. Animals were inoculated with wild-type B16 cells by i.v. injection into the tail vein. Mice were treated with thioridazine (10 and 15 mg/kg x3/week i.p. or 15, and 25 mg/kg/day p.o.) and control animals received saline. Mice were monitored for 21-30 days. Body weight was recorded. After autopsy, the lung weight and number of pulmonary melanoma colonies were determined. Thioridazine administration (i.p. or p.o.) resulted in the reduction of lung tumor burden and an increase in mice survival. In conclusion, several phenothiazines, and particularly thioridazine, induced apoptosis of B16 melanoma cells and demonstrated in vivo anti-tumor activity.

Inhibitory effects ofN-(4-hydrophenyl) retinamide on liver cancer and malignant melanoma cells

To investigate the effect of N-(4-hydrophenyl) retinamide (4-HPR), the derivative of retinoic acid, on inhibition of migration, invasion, cell growth, and induction of apoptosis in hepatocellular carcinoma cells (HCCs) and malignant melanoma cells. 4-HPR was chemically synthesized. Cellular migration and invasion were assayed by Borden chamber experiment. Cell growth was assayed by MTT chromometry. Apoptosis effect was measured using Hoechst 32258 staining and flow cytometry. Gene transfection was performed with lipofectamine. We observed that the migration of HCC and melanoma cells was significantly suppressed by 4-HPR and the migration cells were reduced to 58+/-5.03 (control 201+/-27.2, P<0.05, n = 4) in SMMC 7721-k3 HCC, and to 254+/-25.04 (control 302+/-30.1, P<0.05, n = 4) in melanoma cells after 6-h incubation with 4-HPR. The invasion through reconstituted basement membrane was also significantly reduced by 4-HPR treatment to 11.2+/-3.3 in SMMC 7721-k3 HCC (control 27+/-13.1), and to 24.3+/-3.2 in melanoma cells (control 67.5+/-10.1, P<0.05, n = 3). Cell growth, especially in melanoma cells, was also significantly inhibited. Furthermore, 3 micromol/L of 4-HPR induced apoptosis in B16 melanoma cells (37.11+/-0.94%) more significantly than all-trans retinoic acid (P<0.05), but it failed to induce apoptosis in SMMC 7721-k3 HCC. The mechanism for 4-HPR-induced apoptosis was not clear, but we observed that 4-HPR could regulate p27(kip1), and overexpression of cerebroside sulfotransferase (CST) diminished the apoptosis induced by 4-HPR in melanoma cells. 4-HPR is a potent inhibitor of HCC migration and inducer of melanoma cell apoptosis. CST and p27(kip1) expression might be associated with 4-HPR-induced apoptosis.

Combined effects of Cordyceps sinensis and methotrexate on hematogenic lung metastasis in mice.

We investigated antitumor effects of water extracts of Cordyceps sinensis (WECS). WECS (100 microg/ml) induced apoptosis of B16 melanoma cells after 48 h exposure in vitro as determined by both the TUNEL (TdT-mediated dUTP-biotin nick end labeling) method and the detection of a DNA ladder. In vivo, combined treatment with WECS and methotrexate (MTX) in mice intravenously inoculated with B16 melanoma cells was conducted. Although MTX caused a significant and severe decrease in body weight compared with that in control mice starting 16 days after the start of administration, the mice given both MTX and WECS did not show a significant decrease in body weight. The mean survival time (days) of the control mice, MTX-treated mice (15 mg/kg/day, s.c.), and WECS-treated mice (200 mg/kg/day, p.o.) was 25.0 +/- 0.3, 25.6 +/- 1.3, and 25.7 +/- 1.0 (mean +/- S.E.M. of 6-7 mice), respectively. On the other hand, mean survival time (days) of mice given the combination of MTX and WECS was 28.2 +/- 0.7, significantly longer than the control value. WECS might be beneficial in the prevention of tumor metastasis as an adjuvant agent in cancer chemotherapy.

Flavonoids inhibit cell growth and induce apoptosis in B16 melanoma 4A5 cells.

We investigated the growth inhibitory activity of several flavonoids, including apigenin, luteolin, kaempherol, quercetin, butein, isoliquiritigenin, naringenin, genistein, and daizein against B16 mouse melanoma 4A5 cells. Isoliquiritigenin and butein, belonging to the chalcone group, markedly suppressed the growth of B16 melanoma cells and induced cell death. The other flavonoids tested showed little growth inhibitory activity and scarcely caused cell death. In cells treated with isoliquiritigenin or butein, condensation of nuclei and fragmentation of nuclear DNA, which are typical phenomena of apoptosis, were observed by Hoechst 33258 staining and by agarose gel electrophoresis of DNA. Flowcytometric analysis showed that isoliquiritigenin and butein increased the proportion of hypodiploid cells in the population of B16 melanoma cells. These results demonstrate that isoliquiritigenin and butein inhibit cell proliferation and induce apoptosis in B16 melanoma cells. Extracellular glucose decreased the proportion of hypodiploid cells that appeared as a result of isoliquiritigenin treatment. p53 was not detected in cells treated with either of these chalcones, however, protein of the Bcl-2 family were detected. The level of expression of Bax in cells treated with either of these chalcones was markedly elevated and the level of Bcl-XL decreased slightly. Isoliquiritigenin did not affect Bcl-2 expression, but butein down-regulated Bcl-2 expression. From these results, it seems that the pathway by which the chalcones induce apoptosis may be independent of p53 and dependent on proteins of the Bcl-2 family. It was supposed that isoliquiritigenin induces apoptosis in B16 cells by a mechanism involving inhibition of glucose transmembrane transport and promotion of Bax expression. On the other hand, it was suggested that butein induces apoptosis via down-regulation of Bcl-2 expression and promotion of Bax expression. This mechanism differs from the isoliquiritigenin induction pathway.

Phloretin-induced apoptosis in B16 melanoma 4A5 cells by inhibition of glucose transmembrane transport

Phloretin, a naturally occurring dihydrochalcone, is known to inhibit tumor cell growth in vitro and in vivo. To clarify the anti-tumor effects of phloretin, its apoptosis-inducing effects in B16 melanoma 4A5 cells were examined. Phloretin induced the internucleosomal DNA fragmentation typical of apoptosis in B16 melanoma cells. The addition of extracellular glucose remarkably inhibited the phloretin-induced apoptosis in the cells. When apoptosis was strongly induced in the B16 cells by phloretin, protein kinase C activity was inhibited in the cells. Our results suggest that phloretin induced apoptosis in B16 melanoma 4A5 cells mainly through the inhibition of glucose transmembrane transport. Inhibition of protein kinase C activity by phloretin probably promotes the ratio of apoptotic cells in the cells.

Saikosaponin b2-Induced Apoptosis of Cultured B16 Melanoma Cell Line through Down-regulation of PKC Activity

Saikosaponin (SS) b2was found to inhibit the proliferation of B16 melanoma cells. To explore this mechanism, we employed flow cytometry to determine the distribution of DNA content. The cell cycle of B16 melanoma cells was accumulated in the G1phase followed by induction of apoptosis. This suggests that SSb2-induced proliferation inhibition is caused by G1phase accumulation and that apoptosis induction is G1-phase-accumulation dependent. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, did not interfere with the proliferation and did not induce apoptosis of B16 melanoma cells by itself. However, PMA significantly abolished these effects of SSb2in including proliferation inhibition and apoptosis induction. Down-regulation of the PKC activity may be involved in the effect of SSb2.