Signalling responses linked to betulinic acid‐induced apoptosis are antagonized by MEK inhibitor U0126 in adherent or 3D spheroid melanoma irrespective of p53 status

Abstract

MEK1/2 inhibitors like U0126 can potentiate or antagonize the antitumor activity of cytotoxic agents such as cisplatin, paclitaxel or vinblastine, depending on the drug or the target cells. We now investigated whether U0126, differentially regulates melanoma signaling in response to UV radiation or betulinic acid, a drug lethal against melanoma. This report shows that U0126 inhibits early response (ERK) kinase activation and cyclin A expression in wt p53 C8161 melanoma exposed to either UV radiation or betulinic acid. However, U0126 does not protect from UV damage, but counteracts betulinic acid-mediated apoptosis in the same cells. Protection from the latter drug by joint treatment with U0126 was also evident in wt p53 MelJuso melanoma and mutant p53 WM164 melanoma. The latter cells were the most responsive to betulinic acid, showing a selective decline in the cdk4 protein, without a comparable change in other key cell cycle proteins like cdc2, cdk2, cdk7 or cyclin A, prior to apoptosis-associated PARP fragmentation. Laser scanning cytometry also showed that betulinic acid induced a significant increase in chromatin condensation in WM164 melanoma irrespective of whether they were in adherent form or as multicellular spheroids. All these betulinic acidinduced changes were counteracted by U0126. Our data show for the first time that (a) cdk4 protein is an early target of betulinic acid-induced apoptosis and (b) unrestricted ERK signaling favours betulinic acid-induced apoptosis, but this is counteracted by U0126, partly through counteracting chromatin condensation and restoring Akt activation decreased by betulinic acid treatment. ' 2005 Wiley-Liss, Inc.Key words: laser scanning cytometry; cdk4; G2; ERK/Akt survival cross talk; apoptosis Constitutive activation of p42/p44 MAPK (mitogen-activated protein) kinases or early response (ERK) kinases occurs in tumor progression in prostate carcinoma and melanoma. 1,2 On the basis of the importance of constitutive ERK activation in tumors, pharmacological inhibitors of MAPK kinases MEK1/2 that activate MAPK/ERK have attracted attention since some of them inhibit ERK activation and block human colon carcinoma growth in vivo. 3,4 Pharmacological inhibition of the MAPK kinases MEK1/2 was recently reported as a useful strategy for killing melanoma cells. 5 However, others have shown that the MEK inhibitor U0126 can normalize the morphology of adherent Ki-ras-transformed rat fibroblasts, 6 inhibit invasion of A375 melanoma through Matrigel 7 and promote B16 melanoma differentiation, 8 without exerting a cytotoxic effect on these tumor cells. Moreover, pharmacological inhibition of ERK activation by U0126 or by PD98059 was reported to strongly attenuate apoptosis induced by high dose etoposide, adryamicin, or UV radiation. 9 Enforced activation of ERK by overexpression of MEK-1/Q56P also sensitized cells to DNA damage-induced apoptosis. 9 In contrast, in vivo studies involving injection of TPras melanoma cells into SCID mice resulted in the development of invasive tumors. App...

ERK activation increases nitroprusside induced apoptosis in human melanoma cells irrespective of p53 status: Role of superoxide dismutases

“…To asses this, we used laser scanning cytometry as previously indicated. 5 This confirmed that UO126 induced G 1 accumulation and decreased S + G 2 progression (Fig. 3A).…”

“…[4][5][6][7] Most anti-cancer treatments cause DNA damage, known to promote ERK activation and induce the tumor suppressor wt p53 protein. 8 Mutant p53 protein is frequently found in human cancer 9 and its expression interferes with p53-independent apoptotic pathways.…”

“…19 We now investigated the anti-tumor effect of SNP, an extremely potent vasodilator used during clinical anaesthesia, which behaves as a donor of nitric oxide, cyanide and iron, [20][21][22] or acts mostly through the release of iron after its light inactivation. 22 For these experiments, we used parental wt p53 human C8161 melanoma, 5 and C8161 melanoma variants transduced with mutant p53 R175H or with empty vector. 23 Finally, since SNP is believed to induce oxidative stress 22 we also investigated in C8161 melanoma with differing p53 genotype, whether SNP influences expression of Cu,Zn-SOD, a major defense against reactive oxygen species (ROS) through detoxifying the superoxide anion.…”

Targeting survival cascades induced by activation of Ras/Raf/MEK/ERK, PI3K/PTEN/Akt/mTOR and Jak/STAT pathways for effective leukemia therapy

“…For example, combining MEK inhibitors with betulinic acid, a drug lethal for melanoma cells, antagonized the effects that betulinic acid normally has on apoptosis. 140 Furthermore, the precise timing of the addition of two drugs is important as they may differentially affect cell cycle progression; therefore, one drug may need to be added before the other for a synergynistic response to be observed and perhaps to prevent an antagonistic response. [139][140][141] Combinations of the mTOR inhibitor rapamycin and the cell cycle check point kinase (Chk1) inhibitor UCN-01 also resulted in a synergistic induction of apoptosis in human leukemic cells that was regulated by the Raf/MEK/ERK, Akt and JNK signal transduction pathways.…”

Impairment of lysosomal integrity by B10, a glycosylated derivative of betulinic acid, leads to lysosomal cell death and converts autophagy into a detrimental process

“…PI3K, NF-kB and lipid metabolism are modulated by BA-related compounds, but whether and how these effects contribute to cell death is unknown. [7][8][9][10] A better understanding of the mechanism of action of these compounds is critical to promote their therapeutic usage. Therefore, we investigated in the present study the mode of action of B10, a new glycosylated derivative of BA presenting enhanced cytotoxic activity.…”