Deciphering the dual role and prognostic potential of PINK1 across cancer types.

Metabolic rewiring and deregulation of the cell cycle are hallmarks shared by many cancers. Concerted mutations in key tumor suppressor genes, such as PTEN, and oncogenes predispose cancer cells for marked utilization of resources to fuel accelerated cell proliferation and chemotherapeutic resistance. Mounting research has demonstrated that PTEN-induced putative kinase 1 (PINK1) acts as a pivotal regulator of mitochondrial homeostasis in several cancer types, a function that also extends to the regulation of tumor cell proliferative capacity. In addition, involvement of PINK1 in modulating inflammatory responses has been highlighted by recent studies, further expounding PINK1's multifunctional nature. This review discusses the oncogenic roles of PINK1 in multiple tumor cell types, with an emphasis on maintenance of mitochondrial homeostasis, while also evaluating literature suggesting a dual oncolytic mechanism based on PINK1's modulation of the Warburg effect. From a clinical standpoint, its expression may also dictate the response to genotoxic stressors commonly used to treat multiple malignancies. By detailing the evidence suggesting that PINK1 possesses distinct prognostic value in the clinical setting and reviewing the duality of PINK1 function in a context-dependent manner, we present avenues for future studies of this dynamic protein.

PINK1 depletion sensitizes non-small cell lung cancer to glycolytic inhibitor 3-bromopyruvate: Involvement of ROS and mitophagy.

BACKGROUND: Despite significant strides in understanding the pathophysiology of non-small cell lung cancer (NSCLC), these neoplasms typically present with intrinsic chemo- and radiotherapeutic resistance. Transcriptomic analyses of patient NSCLC tumors stratified by survival times have identified the PTEN-induced putative kinase 1 (PINK1) as a molecular governor of tumor aggressiveness and patient survival time. PINK1 has been shown to confer neuroprotection in models of Parkinson Disease by ensuring proper mitochondrial turnover (mitophagy), the upkeep of ATP production and sequestering of reactive oxygen species (ROS). METHODS: We utilized an shRNA against PINK1 and the glycolytic inhibitor 3-BP to assess effects on NSCLC viability via MTS cell viability assay. ATP levels, caspase-9 activation, mitophagy and ROS production were determined with standardly available kits. Cytochrome c cellular localization and phosphorylated parkin levels were determined using an ELISA. RESULTS: Our results demonstrate that PINK1 depletion in the NSCLC cell line A549 via shRNA, reduced cancer cell proliferation, increased cell death, reduced ATP production, inhibited mitophagy and increased ROS and caspase-9-dependent apoptosis. PINK1 depleted cells were more susceptible to the glycolytic inhibitor 3-bromopyruvate (3-BP), which further perturbed ATP production. PINK1 depletion and 3-BP synergistically increased ROS production, caspase-9-dependent apoptosis and additively repressed mitophagy. CONCLUSIONS: These results suggest that PINK1 depletion alters energetic metabolism and confers sensitivity to agents that inhibit glycolysis. Targeting accelerated tumor cell metabolism may prove useful in the clinical setting while sparing non-malignant tissue.

Vascular-endothelial response to IDH1 mutant fibrosarcoma secretome and metabolite: implications on cancer microenvironment.

Isocitrate dehydrogenases (IDHs) are enzymes involved in the production of α-ketoglutarate (αkg) in normal cellular metabolism. Cells with IDH mutations reduce αkg to 2-hydroxyglutarate (2HG), an oncometabolite, and 2HG directly transforms normal cells to malignant cells through histone demethylation and epigenetic dysregulation. However, whether IDH mutations affect cancer stromal cells is elusive, and little is known whether 2HG may impact the tumor microenvironment. We hypothesized that the IDH mutant cancer secretome and metabolites would stimulate primitive vascular-endothelial genesis. The secretome of IDH1 mutant human fibrosarcoma cells was harvested following medium starvation and was used to treat vascular-endothelial cells using a tube formation assay. GSK864, an allosteric IDH1 inhibitor, was supplemented to the fibrosarcoma secretome to determine its effects on vascular-endothelial tube formation. Exogenous 2HG or as supplemented in the GSK864-treated secretome was applied to further induce vascular-endothelial perturbation. Total vascular-endothelial tube lengths were quantified using NIH/Image J. Two-sided Student's t-tests and Mann-Whitney U tests were used for statistical analysis. The IDH1 mutant fibrosarcoma secretome stimulated vascular-endothelial tube formation by ~138% relative to control. Remarkably, GSK864 attenuated vascular-endothelial tube formation by ~36%, but 2HG not only reversed GSK864 attenuation of tube formation, but also significantly stimulated vascular-endothelial tubes in the GSK864-treated fibrosarcoma secretome. Importantly, 2HG alone augmented vascular-endothelial tube formation that was equivalent to the fibrosarcoma secretome. Thus, 2HG stimulates vascular-endothelial genesis in conjunction with the fibrosarcoma secretome, despite pre-emptive inhibition of IDH1 mutation with GSK864, suggesting that 2HG enables oncogenic angiogenesis via paracrine signaling. Stimulation of vascular-endothelial genesis by 2HG alone, independent of the cancer secretome, suggests that 2HG also activates oncogenic angiogenic pathways in cancer stromal cells. Thus, the IDH mutant cancer secretome stimulates primitive oncogenic angiogenesis through 2HG and/or paracrine pathways. Taken together, these findings suggest novel mechanisms by which the IDH mutant cancer secretome and/or metabolite, specifically 2HG, interacts with the tumor microenvironment by inducing oncogenic angiogenesis in favor of metastasis.

Studies examining the synergy between Dihydrotanshinone and Temozolomide against MGMT+ glioblastoma cells in vitro: Predicting interactions with the blood-brain barrier.

We showed previously that Dihydrotanshinone (DHT) augments temozolomide (TMZ) efficacy by inducing reactive oxygen species production in an in vitro model. Here, the underlying basis of the synergistic effect and the ability of DHT to potentially pass the blood brain barrier (BBB) is investigated using an in vitro model. Trypan blue exclusion assays were used to determine effects of DHT/TMZ combinatorial treatment on GBM cell viability. ELISA was utilized to determine effects on NFkB levels after singular and combinatorial treatment. An in vitro model of the BBB was constructed to predict the potential of DHT to penetrate the BBB in vivo. DHT and TMZ synergistically reduce cancer cell viability, NFkB activity, and markedly halt cell cycle progression. This regimen was also shown to exert minimal effects on astrocytes. Finally, DHT was shown to have the potential of passing through the BBB to a similar extent as TMZ and that paclitaxel's oncolytic effects are completely ablated in the presence of our in vitro BBB. Our data confirms the synergistic interaction between DHT and TMZ and also highlights the potential of combination treatment to sequester NFkB activity and inhibit cell cycle progression. The encouraging data with the BBB model show that the DHT/TMZ combination may be clinically useful and warrants future in vivo testing.

Probing the Oncolytic and Chemosensitizing Effects of Dihydrotanshinone in an In Vitro Glioblastoma Model.

BACKGROUND: Temozolomide is the primary chemotherapeutic agent used to treat glioblastoma. However, many tumors are initially resistant to or develop resistance to temozolomide, mainly due to high levels of O6-methylguanine DNA transferase (MGMT) which repairs DNA damage traditionally caused by temozolomide. Dihydrotanshinone (DHT) is extracted from Salvia miltiorrhiza, a Chinese medicinal plant, and has also been shown to have antiproliferative effects on various cancer cell lines. DHT has been to shown to induce apoptosis via induction endoplasmic reticulum stress, that can reportedly sensitize cells to temozolomide. MATERIALS AND METHODS: MTS cellular proliferation assays or trypan blue viability assays were used to determine the effects of DHT/temozolomide combinatorial treatment. Enzyme-linked immunosorbent assay (ELISA) was used to determine effects on MGMT and P-glycoprotein levels after singular and combinatorial treatment. RESULTS: DHT had a synergistic oncolytic effect in a MGMT-deficient cell line and a sensitizing effect in a MGMT-expressing cell line. Cytotoxicity due to DHT was shown to be reactive oxygen species-dependent, while the combinatorial effect of DHT and temozolomide synergistically reduced MGMT and P-glycoprotein levels. CONCLUSION: DHT was shown to augment temozolomide efficacy, indicating that, since DHT can penetrate the blood-brain barrier, temozolomide in combination with DHT may represent a promising therapeutic option for glioblastoma.

Probing the Molecular Mechanisms Governing the Oncolytic Activity of Paeonia suffruticosa on Triple-negative Breast Cancer Cells In Vitro.

BACKGROUND/AIM: Extracts of Paeonia suffruticosa are traditionally used in Chinese medicine to increase blood flow. Recently, this extract has been shown to possess anti-tumor and anti-inflammatory properties, though this mechanism remains unknown. In the current work, we prepared extracts of P. suffruticosa and analyzed their effects on MDA-MB-231 triple-negative breast cancer cells. MATERIALS AND METHODS: Varying concentrations of an aqueous extract of P. suffruticosa was administered to MDA-MB-231. An MTS assay was used to determine the cell viability. Cytokine production was investigated through enzyme-linked immunosorbent assay (ELISA). Caspase-Glo assays were performed to measure caspase 3/7, 8 and 9 to analyze anti-apoptotic effects. RESULTS: MTS assay for cell viability revealed that the extract increased viability at low concentrations (0.6 mg/ml) and decreased viability observed at concentrations ≥2.5 mg/ml (p<0.01). ELISA for IL-6, IL-2, and TNF-alpha revealed a biphasic dose-response inversely related to viability (p<0.05). IL-24 expression also increased at 2.5 mg/ml and 4.0 mg/ml (p<0.05). Bax levels remained relatively constant while Bcl-2 decreased significantly in all concentrations (p<0.01). Small decreases in Fas ligand levels was observed in parallel with a lack of increase in caspase-8 activity. Most notable was that while 4mg/ml of P. suffruticosa extract reduced MDA-MB-231 viability by >60% (p<0.01), the same concentration reduced the viability of non-transformed HaCat cells by ~8% (p>0.05), suggesting a selective oncolytic effect. CONCLUSION: P. suffruticosa extract has the ability to modulate the production of several tumor suppressive cytokines, induce intrinsic apoptosis and has the capability of reducing cancer burden while sparing healthy tissue.

Lifeguard inhibition of Fas-mediated apoptosis: A possible mechanism for explaining the cisplatin resistance of triple-negative breast cancer cells.

Triple-negative breast cancer does not express estrogen receptor-α, progesterone or the HER2 receptor making hormone or antibody therapy ineffective. Cisplatin may initiate p73-dependent apoptosis in p53 mutant cell lines through Fas trimerization and Caspase-8 activation and Bax up regulation and subsequent Caspase-9 activation. The triple-negative breast cancer, MDA-MB-231, overexpresses the protein Lifeguard, which inhibits Fas-mediated apoptosis by inhibiting Caspase-8 activation after Fas trimerization. The relationship between Fas, Lifeguard and cisplatin is investigated by down regulating Lifeguard via shRNA. Results demonstrate that cisplatin's efficacy increases when Lifeguard is down regulated. Lifeguard Knockdown MDA-MB-231 continue to decrease in cell viability from 24 to 48h after cisplatin treatment while no additional decrease in viability is observed in the Wild-Type MDA over the same period. Higher Caspase-8 activity in the Lifeguard knockdown MDA after cisplatin administration could explain the significant decrease in cell viability from 24 to 48h. This cell type is also more sensitive to Fas ligand-mediated reductions in cell viability, confirming Lifeguard's anti-apoptotic function through the Fas receptor. This research suggests that the efficacy of chemotherapy acting through the Fas pathway would increase if Lifeguard were not overexpressed to inhibit Fas-mediated apoptosis.

Stimulation of cell proliferation and expression of matrixmetalloproteinase-1 and interluekin-8 genes in dermal fibroblasts by copper.

Copper is essential to wound healing as well as a widespread environmental pollutant, with skin aging potential. Wound healing and skin aging are facilitated by matrixmetalloproteinases (MMP), which remodel the extracellular matrix, and interleukin-8 (IL-8), linked with copper. This research investigated the mechanism to copper's role in wound healing or skin aging by regulation of MMP-1 and IL-8 genes. It examined the dose-responsive effects of copper on MMP-1, -2, and -9 activities; MMP-1 and IL-8 gene regulation at protein, mRNA, and promoter levels; tissue inhibitor of matrixmetalloproteinases-1 (TIMP-1) expression; and cell proliferation. Copper stimulated cell proliferation and the expression of MMP-1 and IL-8 genes at the protein, mRNA, and promoter levels, indicating transcriptional regulation, without significantly altering TIMP-1. The research suggests that copper facilitates wound healing as well as skin aging via the induction of MMP-1 expression, with limiting MMP effect at the higher concentrations through enhanced IL-8 expression, which favors extracellular matrix deposition.

Beneficial regulation of matrixmetalloproteinases and their inhibitors, fibrillar collagens and transforming growth factor-beta by Polypodium leucotomos, directly or in dermal fibroblasts, ultraviolet radiated fibroblasts, and melanoma cells.

The extracellular matrix (ECM) that gives tissue its structural integrity is remodeled in skin aging/photoaging and cancer via the increased expression/activities of matrixmetalloproteinases (MMP), inhibition of the tissue inhibitors of matrix metalloproteinases (TIMP), or inhibition of collagen synthesis. Transforming growth factor-beta (TGF-beta), a predominant regulator of the ECM, is inhibited in aging/photoaging and stimulated in carcinogenesis. P. leucotomos (fern) extract has potential to counteract these alterations via its antioxidant, anti-inflammatory and photoprotective properties. The goal of this research was to determine the efficacy of P. leucotomos to (a) directly inhibit MMP-1, 2, 3, and 9 activities, (b) inhibit MMP-2, and stimulate TIMPs, fibrillar collagens and TGF-beta in non-irradiated or ultraviolet (UV) radiated fibroblasts, and (c) inhibit MMPs and TGF-beta, and stimulate TIMPs in melanoma cells. To this purpose, we examined the direct effect of P. leucotomos (0-1%) on MMPs' activities, and its effects on the expression (protein and/or transcription levels) of (1) MMPs and TIMPs in dermal fibroblasts, and melanoma cells, (2) TGF-beta in non-irradiated, UVA (2.5 J/cm2) or UVB (2.5 mJ/cm2) irradiated fibroblasts, and melanoma cells, and (3) types I, III, and V collagen in non-irradiated or UV irradiated fibroblasts. P. leucotomos directly inhibited the activities of MMPs as well as the expression of MMPs in fibroblasts, and melanoma cells while stimulating the expression of TIMPs in these cells. P. leucotomos stimulated types I, III, and V collagen in non-irradiated fibroblasts, and types I and V collagen in UV radiated fibroblasts. P. leucotomos had predominant stimulatory effects on TGF-beta expression in non-irradiated or UV radiated fibroblasts, and inhibited TGF-beta expression in melanoma cells. The effects of P. leucotomos were largely similar to that of ascorbic acid. P. leucotomos demonstrated dual protective effects on the ECM via its inhibition of the ECM proteolytic enzymes and the stimulation of the structural ECM collagens. The effects of P. leucotomos on fibroblasts and melanoma cells may be partly via its cell-specific regulation of TGF-beta expression and partly via its antioxidant property. The intake or topical application of P. leucotomos may be beneficial to skin health, in aging and cancer prevention or treatment.