The mitochondrial cyclophilin D/p53 complexation mediates doxorubicin-induced non-apoptotic death of A549 lung cancer cells

Doxorubicin has displayed significant cytotoxic effects against the lung cancer cells; however, the underlying mechanisms remain inconclusive. In the current study, we provided evidence to show that mitochondrial p53 and cyclophilin D (Cyp-D) complexation is required for doxorubicin-induced death of lung cancer A549 cells. Doxorubicin induced both apoptotic and non-apoptotic death of A549 cells. Cyclosporine A (CsA), the Cyp-D inhibitor, and Cyp-D silencing were prevented doxorubicin-induced non-apoptotic death of A549 cells, while cells overexpressing Cyp-D were hyper-sensitive to doxorubicin. In A549 cells, doxorubicin-activated p53, the latter translocated to mitochondria and physically interacted with Cyp-D. The p53/Cyp-D mitochondrial complexation was prevented by CsA or Cyp-D silencing, or by p53 inhibitor pifithrin-α. Significantly, doxorubicin-induced anti-tumor ability in vivo was also compromised by CsA, or when Cyp-D was silenced. Together, these data suggested that Dox-induced non-apoptotic death of A549 cells requires mitochondrial Cyp-D–p53 complexation.     

Cisplatin-induced non-apoptotic death of pancreatic cancer cells requires mitochondrial cyclophilin-D-p53 signaling

The pancreatic cancer remains a fatal disease for the majority of patients. Cisplatin has displayed significant cytotoxic effects against the pancreatic cancer cells, however the underlying mechanisms remain inconclusive. Here, we found that cisplatin mainly induced non-apoptotic death of the pancreatic cancer cells (AsPC-1 and Capan-2), which was associated with a significant p53 activation (phosphorylation and accumulation). Further, activated p53 was found to translocate to mitochondria where it formed a complex with cyclophilin D (Cyp-D). We provided evidences to support that mitochondrial Cyp-D/p53 complexation might be critical for cisplatin-induced non-apoptotic death of pancreatic cancer cells. Inhibition of Cyp-D by its inhibitor cyclosporine A (CsA), or by shRNA-mediated knockdown suppressed cisplatin-induced pancreatic cancer cell death. Both CsA and Cyp-D knockdown also disrupted the Cyp-D/p53 complex formation in mitochondria. Meanwhile, the pancreatic cancer cells with p53 knockdown were resistant to cisplatin. On the other hand, HEK-293 over-expressing Cyp-D were hyper-sensitive to cisplatin. Interestingly, camptothecin (CMT)-induced pancreatic cancer cell apoptotic death was not affected CsA or Cyp-D knockdown. Together, these data suggested that cisplatin-induced non-apoptotic death requires mitochondria Cyp-D-p53 signaling in pancreatic cancer cells.     

Ultra-violet B (UVB)-induced skin cell death occurs through a cyclophilin D intrinsic signaling pathway

UVB-induced skin cell damage involves the opening of mitochondrial permeability transition pore (mPTP), which leads to both apoptotic and necrotic cell death. Cyclophilin D (Cyp-D) translocation to the inner membrane of mitochondrion acts as a key component to open the mPTP. Our Western-Blot results in primary cultured human skin keratinocytes and in HaCaT cell line demonstrated that UVB radiation and hydrogen peroxide (H(2)O(2)) induced Cyp-D expression, which was inhibited by anti-oxidant N-acetyl cysteine (NAC). We created a stable Cyp-D deficiency skin keratinocytes by expressing Cyp-D-shRNA through lentiviral infection. Cyp-D-deficient cells were significantly less susceptible than their counterparts to UVB- or H(2)O(2)-induced cell death. Further, cyclosporine A (Cs-A), a Cyp-D inhibitor, inhibited UVB- or H(2)O(2)-induced keratinocytes cell death. Reversely, over-expression of Cyp-D in primary keratinocytes caused spontaneous keratinocytes cell death. These results suggest Cyp-D's critical role in UVB/oxidative stress-induced skin cell death.     

Venom present in sea anemone (Heteractis magnifica) induces apoptosis in non-small-cell lung cancer A549 cells through activation of mitochondria-mediated pathway

Lung cancer is a major cause of cancer deaths throughout the world and the complexity of apoptosis resistance in lung cancer is apparent. Venom from Heteractis magnifica caused dose-dependent decreases in survival of the human non-small-cell lung cancer cell line, as determined by the MTT and Crystal Violet assays. The H. magnifica venom induced cell cycle arrest and induced apoptosis of A549 cells, as confirmed by annexin V/propidium iodide staining. The venom-induced apoptosis in A549 cells was characterized by cleavage of caspase-3 and a reduction in the mitochondrial membrane potential. Interestingly, crude extracts from H. magnifica had less effect on the survival of non-cancer cell lines. In the non-cancer cells, the mechanism via which cell death occurred was through necrosis not apoptosis. These findings are important for future work using H. magnifica venom for pharmaceutical development to treat human lung cancer.     

Targeting lung cancer stem‐like cells with TRAIL gene armed oncolytic adenovirus

Lung cancer stem cell (LCSC) is critical in cancer initiation, progression, drug resistance and relapse. Disadvantages showed in conventional lung cancer therapy probably because of its existence. In this study, lung cancer cell line A549 cells propagated as spheroid bodies (named as A549 sphere cells) in growth factors‐defined serum‐free medium. A549 sphere cells displayed CSC properties, including chemo‐resistance, increased proportion of G0/G1 cells, slower proliferation rate, ability of differentiation and enhanced tumour formation ability in vivo. Oncolytic adenovirus ZD55 carrying EGFP gene, ZD55‐EGFP, infected A549 sphere cells and inhibited cell growth. Tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL) armed oncolytic adenovirus, ZD55‐TRAIL, exhibited enhanced cytotoxicity and induced A549 sphere cells apoptosis through mitochondrial pathway. Moreover, small molecules embelin, LY294002 and resveratrol improved the cytotoxicity of ZD55‐TRAIL. In the A549 sphere cells xenograft models, ZD55‐TRAIL significantly inhibited tumour growth and improved survival status of mice. These results suggested that gene armed oncolytic adenovirus is a potential approach for lung cancer therapy through targeting LCSCs.     

Homocysteine modulates the proteolytic potential of human arterial smooth muscle cells through a reactive oxygen species dependant mechanism

Suberoyl bishydroxamic acid (SBHA) as a histone deacetylase (HDAC) inhibitor has various cellular effects such as cell growth and apoptosis. In the present study, we evaluated the effects of SBHA on the growth and death of A549 lung cancer cells. SBHA inhibited the growth of A549 cells with an IC₅₀ of approximately 50 μM at 72 h in a dose-dependent manner. DNA flow cytometric analysis indicated that SBHA induced a G2/M phase arrest of the cell cycle. This agent also induced apoptosis, as evidenced by sub-G1 cells and annexin V-FITC staining cells. SBHA-induced apoptosis was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ_m), Bcl-2 decrease, Bax increase, and the activation of caspase-3. All of the tested caspase inhibitors significantly rescued some cells from SBHA-induced A549 cell death. However, none of the caspase inhibitors prevented the loss of MMP (ΔΨ_m) induced by SBHA. Intracellular reactive oxygen species (ROS) levels including O ₂ ^?? were increased in 50 μM SBHA-treated A549 cells. None of the caspase inhibitors attenuated ROS levels in these cells. SBHA also elevated the number of glutathione (GSH)-depleted cells in A549 cells, which was reduced by treatment with caspase inhibitors. In conclusion, this is the first report that SBHA inhibited the growth of A549 lung cancer cells via caspase-dependent apoptosis, which was related to GSH depletion rather than changes in ROS level.     

The in vitro and in vivo apoptotic effects of Mahonia oiwakensis on human lung cancer cells

Both the root and stem bark of Mahonia species were popular folk medicines. The plant has several proven biological activities including anti-bacterial, anti-fungal, and anti-inflammatory effects. However, Mahonia has not been studied for its anticancer effects. In the present study, we made extracts from Mahonia oiwakensis (MOE), a selected species in Taiwan, and investigated their effects on various human lung cells. We found that MOE-induced apoptotic death in human A549 non-small-cell lung carcinoma (NSCLC) cells in a dose- and time-dependent manner. Treatment with the extracts also caused an increase in the sub-G1 fraction of cells, chromosome condensation, and DNA fragmentation. The mitochondrial-mediated pathway was implicated in this MOE-induced apoptosis as evidenced by the activation of the caspase cascade, cleavage of poly (ADP-ribose) polymerase (PARP), disruption of mitochondrial membrane potential, and release of cytochrome C. A higher ratio of Bax/Bcl-2 proteins and cleavage of Bid were also observed in MOE-induced cell apoptosis. In A549 tumor-xenografted nude mice, MOE also retarded in vivo proliferation (P < 0.05) and induced apoptosis in tumor cells, as shown by a decrease in Ki-67-positive staining (P < 0.05) and increased transferase-mediated dUTP nick-end labeling (TUNEL)-positive staining (P < 0.05). In conclusion, MOE inhibits the growth of human lung cancer cells in vitro and in vivo, suggesting that it may have therapeutic potential against human lung cancer.     

Curcumin-induced melanoma cell death is associated with mitochondrial permeability transition pore (mPTP) opening

Here we studied the role of mitochondrial permeability transition pore (mPTP) opening in curcumin’s cytotoxicity in melanoma cells. In cultured WM-115 melanoma cells, curcumin induced mitochondrial membrane potential (MPP) decrease, cyclophilin-D (CyPD)-adenine nucleotide translocator 1 (ANT-1) (two mPTP components) mitochondrial association and cytochrome C release, indicating mPTP opening. The mPTP blocker sanglifehrin A (SfA) and ANT-1 siRNA-depletion dramatically inhibited curcumin-induced cytochrome C release and WM-115 cell death. CyPD is required for curcumin-induced melanoma cell death. The CyPD inhibitor cyclosporin A (CsA) or CyPD siRNA-depletion inhibited curcumin-induced WM-115 cell death and apoptosis, while WM-115 cells with CyPD over-expression were hyper-sensitive to curcumin. Finally, we found that C6 ceramide enhanced curcumin-induced cytotoxicity probably through facilitating mPTP opening, while CsA and SfA as well as CyPD and ANT-1 siRNAs alleviated C6 ceramide’s effect on curcumin in WM-115 cells. Together, these results suggest that curcumin-induced melanoma cell death is associated with mPTP opening.     

Oxygen Glucose Deprivation (OGD)/Re-Oxygenation-Induced In Vitro Neuronal Cell Death Involves Mitochondrial Cyclophilin-D/P53 Signaling Axis

Oxidative stress-induced neuronal cell death requires opening of the mitochondrial permeability transition pore. P53 mitochondrial translocation and association with Cyclophilin D (Cyp-D) is required for the pore opening. Here we tested this signaling axis in oxygen glucose deprivation (OGD)/re-oxygenation-induced in vitro neuronal death. Using mitochondrion immunoprecipitation, we found that p53 translocated to mitochondrion and associated with Cyp-D in SH-SY5Y cells exposed to (OGD)/re-oxygenation. Disruption of this complex by Cyp-D inhibitor Cyclosporine A (CsA), or by Cyp-D or p53 deficiency, significantly inhibited OGD/re-oxygenation-induced apoptosis-independent cell death. Conversely, over-expression of Cyp-D in SH-SY5Y cells caused spontaneous cell death, and these cells were more vulnerable to OGD/re-oxygenation. Finally, CsA or Cyp-D RNAi suppressed OGD/re-oxygenation-induced neuronal cell death in primary cultures. Together, our study suggests that OGD/re-oxygenation-induced in vitro cell death involves a mitochondrial Cyp-D/p53 signaling axis.     

Gallic acid-induced lung cancer cell death is accompanied by ROS increase and glutathione depletion

Gallic acid (GA) is generally distributed in a variety of plants and foods, and its various biological effects have been reported. Here, we investigated the effects of GA and/or caspase inhibitors on Calu-6 and A549 lung cancer cells in relation to cell death and reactive oxygen species (ROS). The growths of Calu-6 and A549 cells were diminished with an IC(50) of approximately 30 and 150 μM GA at 24 h, respectively. GA also inhibited the growth of primary human pulmonary fibroblast (HPF) cells with an IC(50) of about 300 μM. GA induced apoptosis and/or necrosis in lung cancer cells, which was accompanied by the loss of mitochondrial membrane potential (MMP, ΔΨ(m)). The percents of MMP (ΔΨ(m)) loss and death cells by GA were lower in A549 cells than in Calu-6 cells. Caspase inhibitors did not significantly rescued lung cancer cells from GA-induced cell death. GA increased ROS levels including O(2) (?-) and induced GSH depletion in both lung cancer cells. Z-VAD (pan-caspase inhibitor) did not decrease ROS levels and GSH depleted cell number in GA-treated lung cancer cells. In conclusion, GA inhibited the growth of lung cancer and normal cells. GA-induced lung cancer cell death was accompanied by ROS increase and GSH depletion.     

Manumycin A inhibits triple-negative breast cancer growth through LC3-mediated cytoplasmic vacuolation death

Therapy resistance can be attributed to acquisition of anti-apoptotic mechanisms by the cancer cells. Therefore, developing approaches that trigger non-apoptotic cell death in cancer cells to compensate for apoptosis resistance will help to treat cancer effectively. Triple-negative breast cancers (TNBC) are among the most aggressive and therapy resistant to breast tumors. Here we report that manumycin A (Man A), an inhibitor of farnesyl protein transferase, reduces cancer cell viability through induction of non-apoptotic, non-autophagic cytoplasmic vacuolation death in TNBC cells. Man A persistently induced cytoplasmic vacuolation and cell death through the expression of microtubule-associated protein 1 light chain 3 (LC3) and p62 proteins along with endoplasmic reticulum (ER) stress markers, Bip and CHOP, and accumulation of ubiquitinated proteins. As inhibitors of apoptosis and autophagy failed to block cytoplasmic vacuolation and its associated protein expression or cell death, it appears that these processes are not involved in the death induced by Man A. Ability of thiol antioxidant, NAC in blocking Man A-induced vacuolation, death and its related protein expression suggests that sulfhydryl homeostasis may be the target of Man A. Surprisingly, normal human mammary epithelial cells failed to undergo cytoplasmic vacuolation and cell death, and grew normally in presence of Man A. In conjunction with its in vitro effects, Man A also reduced tumor burden in vivo in xenograft models that showed extensive cytoplasmic vacuoles and condensed nuclei with remarkable increase in the vacuolation-associated protein expression together with increase of p21, p27, PTEN and decrease of pAkt. Interestingly, Man A-mediated upregulation of p21, p27 and PTEN and downregulation of pAkt and tumor growth suppression were also mimicked by LC3 knockdown in MDA-MB-231 cells. Overall, these results suggest novel therapeutic actions by Man A through the induction of non-apoptotic and non-autophagic cytoplasmic vacuolation death by probably affecting ER stress, LC3 and p62 pathways in TNBC but not in normal mammary epithelial cells.     

Suppression of Lung Tumorigenesis by Leucine Zipper/EF Hand–Containing Transmembrane-1

Background

Leucine zipper/EF hand-containing transmembrane-1 (LETM1) encodes for the human homologue of yeast Mdm38p, which is a mitochondria-shaping protein of unclear function. However, a previous study demonstrated that LETM1 served as an anchor protein for complex formation between mitochondria and ribosome, and regulated mitochondrial biogenesis.

Methodology/Principal Findings

Therefore, we examine the possibility that LETM1 may function to regulate mitochondria and lung tumor growth. In this study, we addressed this question by studying in the effect of adenovirus-mediated LETM1 in the lung cancer cell and lung cancer model mice. To investigate the effects of adenovirus-LETM1 in vitro, we infected with adenovirus-LETM1 in A549 cells. Additionally, in vivo effects of LETM1 were evaluated on K-ras ^LA1 mice, human non-small cell lung cancer model mice, by delivering the LETM1 via aerosol through nose-only inhalation system. The effects of LETM1 on lung cancer growth and AMPK related signals were evaluated. Adenovirus-mediated overexpression of LETM1 could induce destruction of mitochondria of lung cancer cells through depleting ATP and AMPK activation. Furthermore, adenoviral-LETM1 also altered Akt signaling and inhibited the cell cycle while facilitating apoptosis. Theses results demonstrated that adenovirus-LETM1 suppressed lung cancer cell growth in vitro and in vivo.

Conclusions/Significance

Adenovirus-mediated LETM1 may provide a useful target for designing lung tumor prevention and treatment.     

Modulation of mitochondrial permeability transition pore affects multidrug resistance in human hepatocellular carcinoma cells

Multidrug resistance (MDR) is a critical problem in the chemotherapy of cancers. Human hepatocellular carcinoma (HCC) responds poorly to chemotherapy owing to its potent MDR. Chemotherapeutic drugs primarily act by inducing apoptosis of cancer cells, and defects in apoptosis may result in MDR. Mitochondrial permeability transition (mPT) is implicated as an important event in the control of cell death or survival and mPT represents a target for the development of cytotoxic drugs. This study aimed to investigate the effects of selective opener (Atractyloside glycoside, ATR) and inhibitor (Cyclosporine A, CsA) of mitochondrial permeability transition pore (mPTP) on a CDDP-resistant HCC cell line (SK-Hep1 cells). In this study, a stable MDR phenotype characterization of SK-Hep1 cell line (SK-Hep1/CDDP cells) was established and used to investigate the role of mPTP in MDR. Results suggested that ATR accelerated the decrease of mitochondrial membrane potential (ΔΨm), reduced the Bax activity, and increased the apoptosis of SK-Hep1/CDDP cells; while CsA inhibited mPTP opening, reduced and delayed the decline of mitochondrial membrane potential, and increased the Bax activity, leading to increased tolerance of SK-Hep1/CDDP cells to apoptosis induction. However, mPTP activity had no effect on the expression of MDR1 in cells,meanwhile the P-gp translocation to mitochondria was increased, and functionally activated. In conclusion, selective modulation of mPTP can affect MDR in human HCC cells. Therefore, activation of mPTP may provide a new strategy to sensitize cancer cells to chemotherapeutic drugs and to reverse the MDR in cancer cells.     

Caveolin-1 knockdown increases the therapeutic sensitivity of lung cancer to cisplatin-induced apoptosis by repressing Parkin-related mitophagy and activating the ROCK1 pathway

Chemotherapy is the first-line treatment option for patients with lung cancer. However, therapeutic resistance occurs through an incompletely understood mechanism. Our research wants to investigate the influence of Caveolin-1 (Cav-1) on the therapeutic sensitivity of lung cancer in vitro. Results in this study demonstrated that Cav-1 levels were markedly inhibited in A549 lung cancer cells after exposure to cisplatin. Knockdown of caveolin further enhanced cisplatin-triggered cancer death in A549 cells. The functional investigation demonstrated that Cav-1 inhibition amplified the mitochondrial stress signaling induced by cisplatin, as evidenced by the mitochondrial reactive oxygen species burst, cellular metabolic disruption, mitochondrial membrane potential reduction, and mitochondrial caspase-9-related apoptosis activation. At the molecular level, cav-1 augmented cisplatin-mediated mitochondrial damage by inhibiting Parkin-related mitochondrial autophagy. Mitophagy activation effectively attenuated the promotive impact of Cav-1 knockdown on mitochondrial damage and cell death. Furthermore, our data indicated that Cav-1 affected Parkin-related mitophagy by activating the Rho-associated coiled-coil kinase 1 (ROCK1) pathway; inhibition of the ROCK1 axis prevented cav-1 knockdown-mediated cell death and mitochondrial damage. Taken together, our results provide ample data illuminate the necessary action exerted by Cav-1 on affecting cisplatin-related therapeutic resistance. Silencing of Cav-1 inhibited Parkin-related mitophagy, thus amplifying cisplatin-mediated mitochondrial apoptotic signaling. This finding identifies the Cav-1/ROCK1/Parkin/mitophagy axis as a potential target to overcome cisplatin-related resistance in lung cancer cells.     

Three new compounds from Artemisia anomala and their antitumor activities

Three new compounds were isolated from Artemisia anomala, and their structures were determined using HR-ESI-MS, IR, UV, and NMR. The antitumor activities of the three compounds were evaluated in the human lung cancer cell line A549 and the human colorectal cancer cell line HCT116. The results showed that compound 2 significantly inhibited cell viability and proliferation and promoted apoptosis of HCT116 and A549 cells, suggesting that compound 2 may be used for colon and lung cancer treatments in clinical practice.     

Bcl-xL Silencing Induces Alterations in hsa-miR-608 Expression and Subsequent Cell Death in A549 and SK-LU1 Human Lung Adenocarcinoma Cells

Bcl-xL is an anti-apoptotic protein that is frequently found to be overexpressed in non-small cell lung cancer leading to an inhibition of apoptosis and poor prognosis. Recently, the role of miRNAs in regulating apoptosis and cell survival during tumorigenesis has become evident, with cancer cells showing perturbed expression of various miRNAs. In this study, we utilized miRNA microarrays to determine if miRNA dysregulation in bcl-xL silenced lung adenocarcinoma cells could be involved in regulating cell death. Short interfering RNA-based transfection of A549 and SK-LU1 lung adenocarcinoma cells was successful in inducing a reduction in bcl-xL expression levels, resulting in a decrease in cell viability. A total of 10 miRNAs were found to be significantly differentially expressed when compared between siRNA-transfected and non-transfected cells including hsa-miR-181a, hsa-miR-769-5p, hsa-miR-361-5p, hsa-miR-1304 and hsa-miR-608. When overexpression studies on hsa-miR-608 was performed via transfection of miRNA mimics, cell death was found to be induced in A549 and SK-LU1 cells in comparison to untreated cells. This effect was reversed when knockdown studies involving anti-sense inhibitors were introduced. Combination of siRNA based silencing of bcl-xL (siBcl-xL) followed by anti-sense inhibitor transfection led to a decrease in the apoptotic population of A549 and SK-LU1 cells in comparison to cells only treated with siBcl-xL, illustrating the connection between bcl-xL, hsa-miR-608 and cell death. Gene target prediction analysis implicated the PI3K/AKT, WNT, TGF-β, and ERK signaling pathways as targets of bcl-xL induced miRNA alterations. We have demonstrated that bcl-xL silencing in A549 and SK-LU1 cells leads to the occurrence of cell death through the dysregulation of specific miRNAs. This study also provides a platform for anti-sense gene therapy whereby miRNA expression can be exploited to increase the apoptotic properties in lung adenocarcinoma cells.     

Growth-inhibiting and apoptosis-inducing activities of Myricanol from the bark of Myrica rubra in human lung adenocarcinoma A549 cells

Myrica rubra (Lour.) Sieb. Et Zucc. is a myricaceae Myrica plant. It is a subtropical fruit tree in China and other Asian countries. The bark of M. rubra is used in Chinese folk medicine because of its antibacterial, antioxidant, anti-inflammatory, and anticancer activities. However, the mechanisms underlying such activities remain unclear.This study investigated whether or not Myricanol extracted from M. rubra bark elicits anti-cancer effects on human lung adenocarcinoma A549 cells by inducing apoptosis in vivo.Myricanol was extracted from M. rubra bark through system solvent extraction and silica gel layer column separation. The results of tritiated thymidine assay, colony formation assay, and flow cytometry indicated that Myricanol inhibited the growth of A549 cells. The effects of Myricanol on the expression of key apoptosis-related genes in A549 cells were evaluated by quantitative PCR and Western blot analyses.Myricanol significantly inhibited the growth of A549 cells in a dose-dependent manner, with a half maximal inhibitory concentration of 4.85 μg/ml. Myricanol significantly decreased colony formation and induced A549 cell apoptosis. Myricanol upregulated the expression of Caspase-3, Caspase-9, Bax, and p21 and downregulated the expression of Bcl-2 at the mRNA and protein levels. These changes were associated with apoptosis.Based on these results, we propose that Myricanol elicits growth inhibitory and cytotoxic effects on lung cancer cells. Therefore, Myricanol may be a clinical candidate for the prevention and treatment of lung cancer.     

Licarin A induces cell death by activation of autophagy and apoptosis in non-small cell lung cancer cells

Lung cancer has a relatively poor prognosis with a low survival rate and drugs that target other cell death mechanism like autophagy may help improving current therapeutic strategy. This study investigated the anti-proliferative effect of Licarin A (LCA) from Myristica fragrans in non-small cell lung cancer cell lines—A549, NCI-H23, NCI-H520 and NCI-H460. LCA inhibited proliferation of all the four cell lines in a dose and time dependent manner with minimum IC50 of 20.03?±?3.12, 22.19?±?1.37 µM in NCI-H23 and A549 cells respectively. Hence NCI-H23 and A549 cells were used to assess the ability LCA to induce autophagy and apoptosis. LCA treatment caused G1 arrest, increase in Beclin 1, LC3II levels and degradation of p62 indicating activation of autophagy in both NCI-H23 and A549 cells. In addition, LCA mediated apoptotic cell death was confirmed by MMP loss, increased ROS, cleaved PARP and decreased pro-caspase3. To understand the role of LCA induced autophagy and its association with apoptosis, cells were analysed following treatment with a late autophagy inhibitor-chloroquine and also after Beclin 1 siRNA transfection. Data indicated that inhibition of autophagy resulted in reduced anti-proliferative as well as pro-apoptotic ability of LCA. These findings confirmed that LCA brought about autophagy dependent apoptosis in non-small cell lung cancer cells and hence it may serve as a potential drug candidate for non-small cell lung cancer therapy.     

Enhancement of auranofin-induced lung cancer cell apoptosis by selenocystine,a natural inhibitor of TrxR1 in vitro and in vivo

Thioredoxin reductase (TrxR) is overpressed in many human tumors and has a key role in regulating intracellular redox balance. Recently, thioredoxin system has emerged as a valuable target for anticancer drug development. Herein we demonstrate that selenocystine (SeC) could enhance auranofin (AF)-induced A549 human lung adenocarcinoma cell apoptosis in vitro and in vivo through synergetic inhibition of TrxR1. SeC pretreatment significantly enhanced AF-induced loss of mitochondrial membrane potential (Δψ_m) by regulating Bcl-2 family proteins. The combined treatment with SeC and AF also resulted in enhanced intracellular reactive oxygen species (ROS) accumulation, DNA damage, and inactivation of ERK and AKT. Inhibitors of ERK and AKT effectively enhanced combined treatment-induced apoptotic cell death. However, inhibition of ROS reversed the apoptosis induced by SeC and AF, and recovered the inactivation of ERK and AKT, which revealed the importance of ROS in cell apoptosis and regulation of ERK and AKT pathways. Moreover, xenograft lung tumor growth in nude mice was more effectively inhibited by combined treatment with SeC and AF by induction of apoptosis through targeting TrxR1 in vivo. Taken together, our results suggest the strategy to use SeC and AF in combination could be a highly efficient way to achieve anticancer synergism by targeting TrxR1.     

Inhibition of the Growth Factor MDK/Midkine by a Novel Small Molecule Compound to Treat Non-Small Cell Lung Cancer

Midkine (MDK) is a heparin-binding growth factor that is highly expressed in many malignant tumors, including lung cancers. MDK activates the PI3K pathway and induces anti-apoptotic activity, in turn enhancing the survival of tumors. Therefore, the inhibition of MDK is considered a potential strategy for cancer therapy. In the present study, we demonstrate a novel small molecule compound (iMDK) that targets MDK. iMDK inhibited the cell growth of MDK-positive H441 lung adenocarcinoma cells that harbor an oncogenic KRAS mutation and H520 squamous cell lung cancer cells, both of which are types of untreatable lung cancer. However, iMDK did not reduce the cell viability of MDK-negative A549 lung adenocarcinoma cells or normal human lung fibroblast (NHLF) cells indicating its specificity. iMDK suppressed the endogenous expression of MDK but not that of other growth factors such as PTN or VEGF. iMDK suppressed the growth of H441 cells by inhibiting the PI3K pathway and inducing apoptosis. Systemic administration of iMDK significantly inhibited tumor growth in a xenograft mouse model in vivo. Inhibition of MDK with iMDK provides a potential therapeutic approach for the treatment of lung cancers that are driven by MDK.