The V600E mutation in the kinase BRAF is frequently detected in melanomas and results in constitutive activation of BRAF, which then promotes cell proliferation by the mitogen-activated protein ...kinase signaling pathway. Although the BRAFV600E kinase inhibitor vemurafenib has remarkable antitumor activity in patients with BRAFV600E-mutated melanoma, its effects are limited by the onset of drug resistance. We found that exposure of melanoma cell lines with the BRAFV600E mutation to vemurafenib decreased the abundance of antiapoptotic proteins and induced intrinsic mitochondrial apoptosis. Vemurafenib-treated melanoma cells showed increased cytosolic concentration of calcium, a potential trigger for endoplasmic reticulum (ER) stress, which can lead to apoptosis. Consistent with an ER stress-induced response, vemurafenib decreased the abundance of the ER chaperone protein glucose-regulated protein 78, increased the abundance of the spliced isoform of the transcription factor X-box binding protein 1 (XBP1) (which transcriptionally activates genes involved in ER stress responses), increased the phosphorylation of the translation initiation factor eIF2α (which would be expected to inhibit protein synthesis), and induced the expression of ER stress-related genes. Knockdown of the ER stress response protein activating transcription factor 4 (ATF4) significantly reduced vemurafenib-induced apoptosis. Moreover, the ER stress inducer thapsigargin prevented invasive growth of tumors formed from vemurafenib-sensitive melanoma cells in vivo. In melanoma cells with low sensitivity or resistance to vemurafenib, combination treatment with thapsigargin augmented or induced apoptosis. Thus, thapsigargin or other inducers of ER stress may be useful in combination therapies to overcome vemurafenib resistance.
The BRAFV600E inhibitor vemurafenib achieves remarkable clinical responses in patients with BRAF‐mutant melanoma, but its effects are limited by the onset of drug resistance. In the case of ...resistance, chemotherapy can still be applied as second line therapy. However, it yields low response rates and strategies are urgently needed to potentiate its effects. In a previous study, we showed that the inhibition of the PI3K‐AKT‐mTOR pathway significantly increases sensitivity of melanoma cells to chemotherapeutic drugs (J. Invest. Dermatol. 2009, 129, 1500). In this study, the combination of the mTOR inhibitor temsirolimus with the chemotherapeutic agent temozolomide significantly increases growth inhibition and apoptosis in melanoma cells compared to temsirolimus or temozolomide alone. The combination of temozolomide with temsirolimus is not only effective in established but also in newly isolated and vemurafenib‐resistant metastatic melanoma cell lines. These effects are associated with the downregulation of the anti‐apoptotic protein Mcl‐1 and the upregulation of the Wnt antagonist Dickkopf homologue 1 (DKK1). Knock‐down of DKK1 suppresses apoptosis induction by the combination of temsirolimus and temozolomide. These data suggest that the inhibition of the mTOR pathway increases sensitivity of melanoma cells towards temozolomide. Chemosensitisation is associated with enhanced expression of the Wnt antagonist DKK1.
Studies indicate that the quality of the doctor–patient relationship moderates the effect of pharmacotherapy. To enhance the quality of the therapeutic relationship in the pharmacotherapy of ...depression, we developed a brief manual with interactive materials for residents in psychiatry and their patients. In a pilot study at a psychiatric university hospital’s outpatient department, we compared patient-centered treatment parameters of a first patient group treated as usual and a second patient group treated using the manual. The study had no influence on the choice of medication. In the manual group, patient satisfaction with the doctor–patient relationship increased significantly at the three-month follow-up. Depression parameters declined in both groups, without group differences. Continuation of antidepressant medication at six months was higher in the manual group. In conclusion, a simple intervention using written materials for doctors prescribing antidepressants improved doctors’ and patients’ satisfaction with treatment.
One of the beneficial effects of peptide-rich protein hydrolysates on diverse pathological conditions has emerged from the concept that their intake as ingredients of dietary supplements or ...pharmaceutical preparations would have the potential to influence the inflammatory response. Leukocytes are physiologically and strategically positioned in the front line of the defense mechanisms and are the main protagonists of the inflammatory response. Their orchestral extravasation through sequential events that lead them to transmigrate across the endothelial barrier is the hallmark of inflammation. As the direct effects of the milk-derived bioactive molecules on circulating blood leukocyte behavior are still little understood and under explored, we have exposed freshly isolated human blood leukocytes to different caseinate-derived phosphopeptides (CPP) produced by trypsin digestion and studied their influence on cellular adhesiveness and mobilization, two of the most crucial leukocyte functions. We also observed their participation on a wound recovery model. The results herein presented and for the first time reported suggest that the CPP obtained by an enzymatic controlled hydrolysis are potential modulators of leukocyte adhesion by altering the expression of VLA-4 and VLA-5 integrins. CPP can also alter leukocyte trafficking by attracting or repelling them influencing, thus, the inflammatory response. In addition, these peptides might favor the recovery of wounds acting positively on fibroblast migration. Our results support the concept that CPP can add benefits for nutritional deficient patients through the enhancement of some crucial leukocyte functions mitigating the inflammatory components often present in malnutrition states.
Farnesyl transferase inhibitors (FTIs) inhibit the farnesylation of proteins, including RAS and RHEB (Ras homolog enriched in brain). RAS signals to the RAF–MEK–ERK (MAPK) and PI3K–AKT–mTOR (AKT) ...signaling pathways, which have a major role in melanoma progression. RHEB positively regulates mammalian target of rapamycin (mTOR). We investigated the effects of the FTI lonafarnib alone and in combination with MAPK (mitogen-activated protein kinase) or AKT (acutely transforming retrovirus AKT8 in rodent T-cell lymphoma) pathway inhibitors on proliferation, survival, and invasive tumor growth of melanoma cells. Lonafarnib alone did not sufficiently inhibit melanoma cell growth. Combinations of lonafarnib with AKT pathway inhibitors did not significantly increase melanoma cell growth inhibition. In contrast, combinations of lonafarnib with MAPK pathway inhibitors yielded additional growth-inhibiting effects. In particular, the combination of the FTI lonafarnib with the pan-RAF inhibitor sorafenib synergistically inhibited melanoma cell growth, significantly enhanced sorafenib-induced apoptosis, and completely suppressed invasive tumor growth in monolayer and organotypic cultures, respectively. Apoptosis induction was associated with upregulation of the endoplasmic reticulum stress-related transcription factors p8 and CHOP (CAAT/enhancer binding protein (C/EBP) homologous protein), and downregulation of the antiapoptotic Bcl-2 (B-cell lymphoma-2) family protein Mcl-1(myeloid cell leukemia 1). Lonafarnib did not affect MAPK and AKT but did affect mTOR signaling. Together, these findings suggest that the FTI lonafarnib inhibits mTOR signaling and enforces sorafenib-induced apoptosis in melanoma cells and may therefore represent an effective alternative for melanoma treatment.
NRAS mutations in malignant melanoma are associated with aggressive disease requiring rapid antitumor intervention, but there is no approved targeted therapy for this subset of patients. In clinical ...trials, the MEK inhibitor (MEKi) binimetinib displayed modest antitumor activity, making combinations a requisite. In a previous study, the BRAF inhibitor (BRAFi) vemurafenib was shown to induce endoplasmic reticulum (ER) stress that together with inhibition of the RAF-MEK-ERK (MAPK) pathway amplified its proapoptotic activity in BRAF-mutant melanoma. The present study investigated whether this effect might extent to NRAS-mutant melanoma, in which MAPK activation would be expected.
BRAFi increased pERK, but also significantly increased growth inhibition and apoptosis induced by the MEKi in monolayer, spheroids, organotypic, and patient-derived tissue slice cultures of NRAS-mutant melanoma. BRAFi such as encorafenib induced an ER stress response via the PERK pathway, as detected by phosphorylation of eIF2α and upregulation of the ER stress-related factors ATF4, CHOP, and NUPR1 and the proapoptotic protein PUMA. MEKi such as binimetinib induced the expression of the proapoptotic protein BIM and activation of the mitochondrial pathway of apoptosis, the latter of which was enhanced by combination with encorafenib. The increased apoptotic rates caused by the combination treatment were significantly reduced through siRNA knockdown of ATF4 and BIM, confirming its critical roles in this process.
The data presented herein encourage further advanced
and clinical studies to evaluate MEKi in combination with ER stress inducing BRAFi as a strategy to treat rapidly progressing NRAS-mutant melanoma.
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Brain metastases are the most common cause of death in patients with metastatic melanoma, and the RAF‐MEK‐ERK and PI3K‐AKT signaling pathways are key players in melanoma progression and drug ...resistance. The BRAF inhibitor vemurafenib significantly improved overall survival. However, brain metastases still limit the effectiveness of this therapy. In a series of patients, we observed that treatment with vemurafenib resulted in substantial regression of extracerebral metastases, but brain metastases developed. This study aimed to identify factors that contribute to treatment resistance in brain metastases. Matched brain and extracerebral metastases from melanoma patients had identical ERK, p‐ERK, and AKT immunohistochemistry staining patterns, but there was hyperactivation of AKT (p‐AKT) and loss of PTEN expression in the brain metastases. Mutation analysis revealed no differences in BRAF, NRAS, or KIT mutation status in matched brain and extracerebral metastases. In contrast, AKT, p‐AKT, and PTEN expression was identical in monolayer cultures derived from melanoma brain and extracerebral metastases. Furthermore, melanoma cells stimulated by astrocyte‐conditioned medium showed higher AKT activation and invasiveness than melanoma cells stimulated by fibroblast‐conditioned medium. Inhibition of PI3K‐AKT signaling resensitized melanoma cells isolated from a vemurafenib‐resistant brain metastasis to vemurafenib. Brain‐derived factors appear to induce hyperactivation of the AKT survival pathway and to promote the survival and drug resistance of melanoma cells in the brain. Thus, inhibition of PI3K‐AKT signaling shows potential for enhancing and/or prolonging the antitumor effect of BRAF inhibitors or other anticancer agents in melanoma brain metastases.
In patients with metastatic melanoma, brain metastases are the most common cause of death. Our findings suggest that hyperactivation of the AKT survival pathway in melanoma brain metastases promotes the survival and drug resistance of melanoma cells in the brain parenchyma. Inhibition of this pathway thus has potential as a novel strategy for the treatment of melanoma brain metastases.
The stem barks of Lafoensia pacari have been traditionally used not only by South Amerindians but also by Brazilian and Paraguayan populations for treating a variety of unhealthy conditions to which ...their biological potential has been scientifically documented in several reports over the last decade. Although its anticancer usage is also popular, no scientific support for such activity has been found.
To provide scientific evidence for the anticancer popularity of Lafoensia pacari.
Extracts prepared according to the popular use along with a methanol extract and its four fractions were produced from Lafoensia pacari stem barks. The chromatogram profile of each one was obtained by HPLC. Several tumor cell lines were exposed to these solutions in in vitro assays and the effects evaluated by morphological, growth, and cell cycle status changes.
High toxicity determined by the lactate dehydrogenase levels with a significant drop in the cell proliferation index were found for all cell lines included in this study after exposition to Lafoensia pacari extract and fractions. The morphological features along with the expression of annexin V have strongly suggested apoptosis induction, which has been confirmed by G0/G1 cell cycle arrest.
The data have clearly shown that exposition of human tumor cell lines to Lafoensia pacari stem barks extract leads to apoptosis induction due to cell cycle arrest in G0/G1 phases, supporting its anticancer use.
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