Attenuation of PI3K-Akt-mTOR Pathway to Reduce Cancer Stemness on Chemoresistant Lung Cancer Cells by Shikonin and Synergy with BEZ235 Inhibitor.

Lung cancer is considered the number one cause of cancer-related deaths worldwide. Although current treatments initially reduce the lung cancer burden, relapse occurs in most cases; the major causes of mortality are drug resistance and cancer stemness. Recent investigations have provided evidence that shikonin generates various bioactivities related to the treatment of cancer. We used shikonin to treat multi-resistant non-small lung cancer cells (DOC-resistant A549/D16, VCR-resistant A549/V16 cells) and defined the anti-cancer efficacy of shikonin. Our results showed shikonin induces apoptosis in these ABCB1-dependent and independent chemoresistance cancer sublines. Furthermore, we found that low doses of shikonin inhibit the proliferation of lung cancer stem-like cells by inhibiting spheroid formation. Concomitantly, the mRNA level and protein of stemness genes (Nanog and Oct4) were repressed significantly on both sublines. Shikonin reduces the phosphorylated Akt and p70s6k levels, indicating that the PI3K/Akt/mTOR signaling pathway is downregulated by shikonin. We further applied several signaling pathway inhibitors that have been used in anti-cancer clinical trials to test whether shikonin is suitable as a sensitizer for various signaling pathway inhibitors. In these experiments, we found that low doses shikonin and dual PI3K-mTOR inhibitor (BEZ235) have a synergistic effect that inhibits the spheroid formation from chemoresistant lung cancer sublines. Inhibiting the proliferation of lung cancer stem cells is believed to reduce the recurrence of lung cancer; therefore, shikonin's anti-drug resistance and anti-cancer stem cell activities make it a highly interesting molecule for future combined lung cancer therapy.

Clinical Outcomes after Intracorporeal versus Extracorporeal Anastomosis in Patients Undergoing Laparoscopic Right Hemicolectomy for Colon Cancer.

Background and Objectives: Laparoscopic right hemicolectomy (LRHC) is commonly performed for patients with colon cancer, selecting between intracorporeal anastomosis (ICA) or extracorporeal anastomosis (ECA). However, the impact of ICA versus ECA on patient outcomes remains debatable. The varying levels of experience among surgeons may influence the outcomes. Therefore, this study sought to compare the short- and long-term outcomes of LRHC using ICA versus ECA. Materials and Methods: This retrospective study extracted patient data from the medical records database of Changhua Christian Hospital, Taiwan, from 2017 to 2020. Patients with colon cancer who underwent LRHC with either ICA or ECA were included. Primary outcomes were post-surgical outcomes and secondary outcomes were recurrence rate, overall survival (OS), and cancer-specific survival (CSS). Between-group differences were compared using chi-square, t-tests, and Fisher's exact tests and Mann-Whitney U tests. Associations between study variables, OS, and CSS were determined using Cox analyses. Results: Data of 240 patients (61 of ICA and 179 of ECA) with a mean age of 65.0 years and median follow-up of 49.3 months were collected. No recognized difference was found in patient characteristics between these two groups. The ICA group had a significantly shorter operation duration (median (IQR): 120 (110-155) vs. 150 (130-180) min) and less blood loss (median (IQR): 30 (10-30) vs. 30 (30-50) mL) than the ECA group (p < 0.001). No significant differences were found in 30-day readmission (ICA vs. ECA: 1.6% vs. 2.2%, p > 0.999) or recurrence (18.0% vs. 13.4%, p = 0.377) between the two groups. Multivariable analyses revealed no significant differences in OS (adjusted hazard ratio (aHR): 0.65; 95% confidence interval (CI): 0.25-1.44) or CSS (adjusted sub-hazard ratio (aSHR): 0.41, 95% CI: 0.10-1.66) between groups. Conclusions: Both ICA and ECA in LRHC for colon cancer had similar outcomes without statistically significant differences in post-surgical complications, 30-day readmission rates, recurrence rate, and long-term survival outcomes. However, ICA may offer two advantages in terms of a shorter operative duration and reduced blood loss.

Curcumin Induces Apoptosis of Chemoresistant Lung Cancer Cells via ROS-Regulated p38 MAPK Phosphorylation.

This study aimed to challenge chemoresistance by curcumin (CUR) with drug-selected human lung cancer A549 sublines that continuously proliferate in the present of docetaxel (DOC) and vincristine (VCR). Their sensitivities to CUR were measured by MTT assay and the particular intracellular reactive oxygen species (ROS) was detected by fluorescence activated cell sorting (FACS) analysis. Apoptosis was analyzed by Annexin V assay of the flow cytometry. Inhibitors and RNA interference were used to examine the signaling pathway regulated by the kinases. The obtained data demonstrated that CUR induces chemoresistant cell apoptosis by generating ROS and application of N-acetylcysteine (NAC) blocks ROS production, resulting in apoptosis suppression. Phosphorylation of extracellular regulated kinase (ERK), p38 MAPK, and eIF-2α were increased but c-Jun N-terminal kinase (JNK) did not increase when chemoresistant cells were treated with CUR. Downregulation of ERK and p38 MAPK phosphorylation by their inhibitors had no effect on CUR-induced apoptosis. Interestingly, the knockdown of p38 MAPK with shRNA significantly reduced CUR-induced apoptosis on the chemoresistant sublines. Phosphorylation of the eIF-2α protein was inhibited when p38 MAPK was knocked down in DOC-resistant A549 cells, but a high level of phosphorylated eIF-2α protein remained on the VCR-resistant A549 cells when p38 MAPK was knocked down. These data confirmed that CUR-augmented ROS potently induced apoptosis via upregulated p38 MAPK phosphorylation. Therefore, activated p38 MAPK is considered a pro-apoptotic signal for CUR-induced apoptosis of chemoresistant human lung cancer cells.

miR-145-5p Targets Sp1 in Non-Small Cell Lung Cancer Cells and Links to BMI1 Induced Pemetrexed Resistance and Epithelial-Mesenchymal Transition.

Pemetrexed is a folic acid inhibitor used as a second-line chemotherapeutic agent for the treatment of locally advanced or metastatic non-small cell lung cancer (NSCLC), which accounts for 85% of lung cancers. However, prolonged treatment with pemetrexed may cause cancer cells to develop resistance. In this study, we found increased expressions of BMI1 (B Lymphoma Mo-MLV insertion region 1 homolog) and Sp1 and a decreased expression of miR-145-5p was found in pemetrexed-resistant A400 cells than in A549 cells. Direct Sp1 targeting activity of miR-145-5p was demonstrated by a luciferase based Sp1 3'-UTR reporter. Changed expression of miR-145-5p in A400 or A549 cells by transfection of miR-145-5p mimic or inhibitor affected the sensitivity of the cells to pemetrexed. On the other hand, the overexpression of Sp1 in A549 cells caused the decreased sensitivity to pemetrexed, induced cell migratory capability, and epithelial-mesenchymal transition (EMT) related transcription factors such as Snail Family Transcriptional Repressor 1 and Zinc Finger E-Box Binding Homeobox 1. In addition, the overexpression of BMI1 in the A549 cells resulted in an increase in Sp1 and a decrease in miR-145-5p accompanied by the elevations of cell proliferation and EMT transcription factors, which could be reduced by the overexpression of miR-145-5p or by treatment with the Sp1 inhibitor of mithramycin A. In conclusion, the results of this study suggest that the downregulation of miR-145-5p by BMI1 overexpression could lead to the enhanced expression of Sp1 to induce the EMT process in pemetrexed-resistant NSCLC cells. These results suggest that increasing miR-145-5p expression by delivering RNA drugs may serve as a sensitizing agent for pemetrexed-resistant NSCLC patients.

Combination treatment of Src inhibitor Saracatinib with GMI, a Ganoderma microsporum immunomodulatory protein, induce synthetic lethality via autophagy and apoptosis in lung cancer cells.

Saracatinib is an oral Src-kinase inhibitor and has been studied in preclinical models and clinical trials of cancer therapy. GMI, a fungal immunomodulatory protein from Ganoderma microsporum, possesses antitumor capacity. The aim of this study is to evaluate the cytotoxic effect of combination treatment with saracatinib and GMI on parental and pemetrexed-resistant lung cancer cells. Cotreatment with saracatinib and GMI induced synergistic and additive cytotoxic effect in A549 and A400 cells by annexin V/propidium iodide assay and combination index. Using western blot assay, saracatinib, and GMI combined treatment synergistically induced caspase-7 activation in A549 cells. Different from A549 cells, saracatinib and GMI cotreatment markedly increased LC3B-II in A400 cells. ATG5 silencing abolished the caspase-7 activation and reduced cell death in A549 cells after cotreatment. This is the first study to provide a novel strategy of treating lung cancer with or without drug resistance via combination treatment with GMI and saracatinib.

Enhancement of cytotoxicity and induction of apoptosis by cationic nano-liposome formulation of n-butylidenephthalide in breast cancer cells.

Breast cancer is the second most common malignancy in women. Current clinical therapy for breast cancer has many disadvantages, including metastasis, recurrence, and poor quality of life. Furthermore, it is necessary to find a new therapeutic drug for breast cancer patients to meet clinical demand. n-Butylidenephthalide (BP) is a natural and hydrophobic compound that can inhibit several tumors. However, BP is unstable in aqueous or protein-rich environments, which reduces the activity of BP. Therefore, we used an LPPC (Lipo-PEG-PEI complex) that can encapsulate both hydrophobic and hydrophilic compounds to improve the limitation of BP. The purpose of this study is to investigate the anti-tumor mechanisms of BP and BP/LPPC and further test the efficacy of BP encapsulated by LPPC on SK-BR-3 cells. BP inhibited breast cancer cell growth, and LPPC encapsulation (BP/LPPC complex) enhanced the cytotoxicity on breast cancer by stabilizing the BP activity and offering endocytic pathways. Additionally, BP and LPPC-encapsulated BP induced cell cycle arrest at the G0/G1 phase and might trigger both extrinsic as well as intrinsic cell apoptosis pathway, resulting in cell death. Moreover, the BP/LPPC complex had a synergistic effect with doxorubicin of enhancing the inhibitory effect on breast cancer cells. Consequently, LPPC-encapsulated BP could improve the anti-cancer effects on breast cancer in vitro. In conclusion, BP exhibited an anti-cancer effect on breast cancer cells, and LPPC encapsulation efficiently improved the cytotoxicity of BP via an acceleration of entrapment efficiency to induce cell cycle block and apoptosis. Furthermore, BP/LPPC exhibited a synergistic effect in combination with doxorubicin.

L-Type Amino Acid Transporter 1 Regulates Cancer Stemness and the Expression of Programmed Cell Death 1 Ligand 1 in Lung Cancer Cells.

The l-type amino acid transporter 1 (LAT1) is a membranous transporter that transports neutral amino acids for cells and is dysregulated in various types of cancer. Here, we first observed increased LAT1 expression in pemetrexed-resistant non-small cell lung cancer (NSCLC) cells with high cancer stem cell (CSC) activity, and its mRNA expression level was associated with shorter overall survival in the lung adenocarcinoma dataset of the Cancer Genome Atlas database. The inhibition of LAT1 by a small molecule inhibitor, JPH203, or by RNA interference led to a significant reduction in tumorsphere formation and the downregulation of several cancer stemness genes in NSCLC cells through decreased AKT serine/threonine kinase (AKT)/mammalian target of rapamycin (mTOR) activation. The treatment of the cell-permeable leucine derivative promoted AKT/mTOR phosphorylation and reversed the inhibitory effect of JPH203 in the reduction of CSC activity in pemetrexed-resistant lung cancer cells. Furthermore, we observed that LAT1 silencing caused the downregulation of programmed cell death 1 ligand 1 (PD-L1) on lung cancer cells. The PD-L1+/LAT1+ subpopulation of NSCLC cells displayed great CSC activity with increased expression of several cancer stemness genes. These data suggest that LAT1 inhibitors can serve as anti-CSC agents and could be used in combination with immune checkpoint inhibitors in lung cancer therapy.

Metabolic Syndrome Prevalence and Cardiovascular Risk Assessment in HIV-Positive Men with and without Antiretroviral Therapy.

Treatment of HIV infection is a lifelong process and associated with chronic diseases. We evaluated the prevalence and predictors of metabolic syndrome (MetS) and cardiovascular diseases (CVDs) with individual antiretroviral drugs exposure among HIV-infected men in Taiwan. A total of 200 patients' data were collected with a mean age of 32.9. Among them, those who had CD4 positive cell number less than 350/mL were eligible to have highly active antiretroviral therapy (HAART). Patients were divided into group-1 that contains 45 treatment-naïve participants, and group-2 that includes 155 HAART treatment-experienced participants. MetS prevalence between group-1 and group-2 was 18% and 31%, respectively. The Framingham Risk Score (FRS) for the naïve and experienced groups were 4.7 ± 4.2 and 3.87 ± 5.92, respectively. High triglyceride (TG > 150 mg/dL) in group-1 and group-2 were 15.6% and 36.6% (p < 0.05), whereas, lower high-density lipoprotein (HDL < 39 mg/dL) in group-1 and group-2 presented as 76.7% versus 51% (p < 0.05), respectively. In group-2, treatment with protease inhibitors (PIs) resulted in higher TG levels when compared with non-nucleotide reverse transcriptase inhibitors (NNRTIs) and integrase inhibitors (InSTIs). The prevalence of MetS in the treatment-naïve group was lower than that of the treatment-experienced group; high TG level resulted in higher MetS prevalence in the treatment-experienced group. In contrast, the cardiovascular risk of FRS in the treatment-naïve group was higher than that of the treatment-experienced group, which may result from the low HDL level. Although group-1 participants have a higher risk of developing CVDs, in group-2, an increasing TG level in PIs user indicated higher CVDs risk. TG and HDL are two significant biofactors that required regular evaluation in HIV-positive individuals.

CD133 inhibition via autophagic degradation in pemetrexed-resistant lung cancer cells by GMI, a fungal immunomodulatory protein from Ganoderma microsporum.

BACKGROUND: Adaptive drug resistance is an unfavourable prognostic factor in cancer therapy. Pemetrexed-resistant lung cancer cells possess high-metastatic ability via ERK-ZEB1 pathway-activated epithelial-mesenchymal transition. GMI is a fungal immunomodulatory protein that suppresses the survival of several cancer cells. METHODS: Cell viability was analysed by MTT, clonogenic, tumour spheroid, and cancer stem cell sphere assays. Western blot assay was performed to detect the protein expression. Chemical inhibitors and ATG5 shRNA were used to inhibit autophagy. Tumour growth was investigated using xenograft mouse model. RESULTS: GMI decreased the viability with short- and long-term effects and induced autophagy but not apoptosis in A549/A400 cells. GMI downregulated the expression levels of CD133, CD44, NANOG and OCT4. GMI induces the protein degradation of CD133 via autophagy. CD133 silencing decreased the survival and proliferation of A549/A400 cells. GMI suppressed the growth and CD133 expression of A549/A400 xenograft tumour. CONCLUSIONS: This study is the first to reveal the novel function of GMI in eliciting cytotoxic effect and inhibiting CD133 expression in pemetrexed-resistant lung cancer cells via autophagy. Our finding provides evidence that CD133 is a potential target for cancer therapy.

Cytotoxicity of naringenin induces Bax-mediated mitochondrial apoptosis in human lung adenocarcinoma A549 cells.

Naringenin (NGEN), a natural flavonoid has growth inhibition and apoptosis-inducing activities in several cancer cells. However, the cytotoxicity mechanisms of NGEN in cell death of lung cancer cells have not been fully defined. In present study, treatment of human lung adenocarcinoma A549 cells with NGEN resulted in time- and dose-dependent decreases in cell viability. Moreover, NGEN significantly induced apoptosis evidenced by morphological changes, DAPI staining, TUNEL assay and sub-G1 population increase. In NGEN-treated cells, intensely upregulated Bax and down-regulated Bcl-2 proteins were detected and the Bax protein associated with the mitochondrial membrane was analyzed by subcellular fractionation. Knockdown of the Bax expression by the shRNA method dramatically protected A549 cells against NGEN-induced apoptosis. Treatment with the inhibitors of caspase-3, -8, or -9 significantly reduced NGEN-induced apoptotic deaths. Taken together, our results demonstrate that NGEN-induced apoptosis may occur via a Bax-activated mitochondrial pathway in lung adenocarcinoma A549 cells.

Pogostemon cablin Triggered ROS-Induced DNA Damage to Arrest Cell Cycle Progression and Induce Apoptosis on Human Hepatocellular Carcinoma In Vitro and In Vivo.

The purpose of the study was to elucidate the anti-hepatoma effects and mechanisms of Pogostemon cablin essential oils (PPa extract) in vitro and in vivo. PPa extract exhibited an inhibitory effect on hepatocellular carcinoma (HCC) cells and was less cytotoxic to normal cells, especially normal liver cells, than it was to HCC cells, exerting a good selective index. Additionally, PPa extract inhibited HCC cell growth by blocking the cell cycle at the G0/G1 phase via p53 dependent or independent pathway to down regulated cell cycle regulators. Moreover, PPa extract induced the FAS-FASL-caspase-8 system to activate the extrinsic apoptosis pathway, and it increased the bax/bcl-2 ratio and reduced ΔΨm to activate the intrinsic apoptosis pathway that might be due to lots of reactive oxygen species (ROS) production which was induced by PPa extract. In addition, PPa extract presented to the potential to act synergistically with sorafenib to effectively inhibit HCC cell proliferation through the Akt/mTOR pathway and reduce regrowth of HCC cells. In an animal model, PPa extract suppressed HCC tumor growth and prolonged lifespan by reducing the VEGF/VEGFR axis and inducing tumor cell apoptosis in vivo. Ultimately, PPa extract demonstrated nearly no or low system-wide, physiological, or pathological toxicity in vivo. In conclusion, PPa extract effectively inhibited HCC cell growth through inducing cell cycle arrest and activating apoptosis in vitro and in vivo. Furthermore, PPa extract exhibits less toxicity toward normal cells and organs than it does toward HCC cells, which might lead to fewer side effects in clinical applications. PPa extract may be developed into a clinical drug to suppress tumor growth or functional food to prevent HCC initiation or chemoprotection of HCC recurrence.

Extract Derived from Cedrus atlantica Acts as an Antitumor Agent on Hepatocellular Carcinoma Growth In Vitro and In Vivo.

Cedrus atlantica is widely used in herbal medicine. However, the anti-cancer activity of C. atlantica extract (CAt extract) has not been clarified in hepatocellular carcinoma. In the study, we elucidated the anti-hepatoma capacity of CAt extract on HCC in vitro and in vivo. To explore the anti-hepatoma mechanisms of the CAt extract in vitro, HCC and normal cells were treated with the CAt extract, which showed marked inhibitory effects on HCC cells in a dose-dependent manner; in contrast, the CAt extract treatment was less cytotoxic to normal cells. In addition, our results indicate that the CAt extract induced apoptosis via caspase-dependent and independent apoptosis pathways. Furthermore, the CAt extract inhibited HCC tumor cell growth by restraining cell cycle progression, and it reduced the signaling of the AKT, ERK1/2, and p38 pathways. In the xenograft model, the CAt extract suppressed HCC tumor cell growth and prolonged lifespan by inhibiting PCNA protein expression, repressing part of the VEGF-induced autocrine pathway, and triggering strong expression of cleaved caspase-3, which contributed to cell apoptosis. Moreover, the CAt extract did not induce any obvious changes in pathological morphology or body weight, suggesting it had no toxicity. CAt extract exerted anti-tumor effects on HCC in vitro and in vivo. Thus, CAt extract could be used as a potential anti-cancer therapeutic agent against HCC.

MicroRNA-21 promotes tumour malignancy via increased nuclear translocation of ß-catenin and predicts poor outcome in APC-mutated but not in APC-wild-type colorectal cancer.

MiR-21 has been associated with poor prognosis in colon adenocarcinomas. However, in our preliminary data, the prognostic value of miR-21 levels was observed only in adenomatous polyposis coli (APC)-mutated tumours, not in APC-wild-type tumours. We explored whether ß-catenin nuclear translocation was synergistically promoted by miR-21 in APC-mutated cells but not in APC-wild-type cells. We enrolled 165 colorectal tumour to determine APC mutation, miR-21 levels and nuclear ß-catenin expression by direct sequencing, real-time PCR and immunohistochemistry. Overall survival and relapse-free survival were analysed by Kaplan-Meier and Cox regression models. The mechanistic action of ß-catenin nuclear translocation modulated by miR-21 and its effect on cell invasion were evaluated in a cell model. Positive nuclear ß-catenin expression was more commonly occurred in APC-mutated tumours than in APC-wild-type tumours. High miR-21 levels were relatively more common in tumours with positive nuclear ß-catenin expression than in those with negative nuclear ß-catenin expression. APC-mutated tumours with high miR-21 levels had shorter overall survival and relapse-free survival periods compared with others. However, the prognostic value of miR-21 levels was not observed in APC-wild-type tumours. Phosphorylation of ß-catenin at Ser552 via the miR-21-mediated PTEN/AKT axis plays a critical role in ß-catenin nuclear translocation in APC-mutated cells but not in APC-wild-type cells. Moreover, nuclear ß-catenin expression increased by miR-21 is responsible for the capability of invasiveness. In summary, nuclear translocation of ß-catenin increased by miR-21 promotes tumour malignancy and a poor outcome in APC-mutated patients but not in APC-wild-type colorectal cancer.

Protective Effect of Curcumin on the Tight Junction Integrity and Cellular Senescence in Human Retinal Pigment Epithelium of Early Diabetic Retinopathy.

Diabetic retinopathy (DR) is a secondary complication of diabetes that can lead to visual impairment and blindness. The retinal pigment epithelium (RPE) is a monolayer of pigment cells that forms the blood-retinal barrier (BRB) via tight junction (TJ) proteins and plays a crucial role in the physiological function of the retina. Hyperglycemia induces RPE death and BRB breakdown, which accelerates the process of DR. Curcumin, an active extract of Curcuma longa , has anti-inflammatory, antioxidant, antiapoptotic, and neuroprotective properties. However, the effect of Curcumin on the BRB under high glucose conditions remains unknown. This study aimed to investigate the protective effects of Curcumin on RPE physiology in vitro and in vivo . Curcumin significantly alleviated cell viability inhibition under high glucose conditions. Moreover, high glucose reduced extracellular signal-regulated kinase and Akt pathways activation to diminish RPE cell growth but reversed by Curcumin treatment. Curcumin protected not only TJ integrity but also retinoid regeneration through TJ proteins and isomerase modulation in diabetic retina. Furthermore, Curcumin decreased the expression of angiogenic factor to inhibit retinal neovascularization. Finally, Curcumin treatment markedly reduced apoptosis during hyperglycemia. In conclusion, Curcumin can alleviate the progression of DR by promoting RPE survival, TJ integrity, retinoid isomerase activity, RPE senescence inhibition, and neovascularization. Therefore, Curcumin exhibits high potential for use as a therapeutic agent for early DR.

HER2 mutations in advanced cervical neuroendocrine carcinoma: implications for trastuzumab deruxtecan therapy.

Recent clinical evidence shows that the antibody-drug conjugate (ADC) trastuzumab deruxtecan (T-DXd) can successfully treat patients with advanced HER2-mutant non-small cell lung cancer (NSCLC). We aimed to characterize HER2 mutations in cervical neuroendocrine carcinoma (NEC) among Taiwanese women to provide the rationale for exploring T-DXd as a tumor-agnostic targeted therapy option. We analyzed 12 archived primary cervical NEC samples from Taiwanese patients. Tumor-rich areas were marked for microdissection on 10 µm unstained sections. DNA was extracted, and HER2 hotspots were sequenced using a targeted panel on the Illumina MiSeq. HER2 missense mutations were identified in 5 of 12 cases (41.7%). Of the 5 cases with mutations, 2 patients (40%) had a single mutation, while 3 patients (60%) had double mutations. We detected 4 substitutions outside the tyrosine kinase domain (non-TKD), which were p.P1170A, p.S305C, p.I655V, and a novel T328K alteration. No mutations were found within the tyrosine kinase domain (TKD). The 41.7% HER2 mutation rate warrants expanded screening and future clinical investigation of the T-DXd targeting HER2 mutations in cervical NEC patients. Overall, this study contributes to the molecular understanding of cervical NEC and lays the groundwork for developing more effective treatment strategies.

Activation of NF-κB by SOD2 promotes the aggressiveness of lung adenocarcinoma by modulating NKX2-1-mediated IKKß expression.

Magnesium superoxide dismutase (SOD2) has been shown to cause dysfunction of p53 transcriptional activity, whereas, in turn, SOD2 expression is regulated by p53 to modulate lung tumorigenesis. In this study, we found that the level of SOD2 expression in a panel of lung cancer cells was negatively correlated with that of NK2 homeobox 1 (NKX2-1) but was not associated with p53 status. Mechanistic studies indicated that a decrease in NKX2-1 caused by SOD2-activated IKKß transcription was achieved by derepression of binding of Sp1 to the IKKß promoter. Immunoprecipitation, glutathione S-transferase pull-down experiments and electrophoretic mobility shift assays demonstrated a direct interaction between NKX2-1 and Sp1, blocking Sp1-mediated IKKß transcription. SOD2-mediated nuclear factor-kappaB activation, via elevation of IKKß transcription, promoted anchorage-independent soft-agar growth, invasion and xenograft tumor formation, because of development of the epithelial-to-mesenchymal transition. The expression level of NKX2-1 messenger RNA was negatively associated with the extent of SOD immunostaining and the IKKß messenger RNA expression level in lung tumors. The extent of SOD2 immunostaining and IKKß messenger RNA levels may independently predict overall survival and relapse-free survival in lung adenocarcinoma patients. In summary, we found that SOD2 activates nuclear factor-kappaB signaling by increasing IKKß transcription, which results in progression of lung adenocarcinoma and poorer patient outcomes. We suggest that IKKß may potentially be targeted to improve outcomes in patients with SOD2-positive tumors.

Pemetrexed induces both intrinsic and extrinsic apoptosis through ataxia telangiectasia mutated/p53-dependent and -independent signaling pathways.

Pemetrexed, a new-generation antifolate, has demonstrated promising single-agent activity in front- and second-line treatments of non-small cell lung cancer. However, the molecular mechanism of pemetrexed-mediated antitumor activity remains unclear. The current study shows that pemetrexed induced DNA damage and caspase-2, -3, -8, and -9 activation in A549 cells and that treatment with caspase inhibitors significantly abolished cell death, suggesting a caspase-dependent apoptotic mechanism. The molecular events of pemetrexed-mediated apoptosis was associated with the activation of ataxia telangiectasia mutated (ATM)/p53-dependent and -independent signaling pathways, which promoted intrinsic and extrinsic apoptosis by upregulating Bax, PUMA, Fas, DR4, and DR5 and activating the caspase signaling cascade. Supplementation with dTTP allowed normal S-phase progression and rescued apoptotic death in response to pemetrexed. Overall, our findings reveal that the decrease of thymidylate synthase and the increase of Bax, PUMA, Fas, DR4, and DR5 genes may serve as biomarkers for predicting responsiveness to pemetrexed.

E6 and E7 of human papillomavirus type 18 and UVB irradiation corporately regulate interleukin-6 and interleukin-8 expressions in basal cell carcinoma.

The lack of a human papillomavirus (HPV)-infected skin cancer cell line has hampered the investigation of the interaction of UV and HPV in skin carcinogenesis. We identified a human basal cell carcinoma (BCC-1/KMC) cell line integrated with E6 and E7 genes of high-risk HPV type 18 and demonstrated that repression of E6 and E7 results in proliferation inhibition. Sublethal ultraviolet-B (UVB) irradiation induced the expressions of interleukin-6 (IL-6) and interleukin-8 (IL-8), as well as viral E6 and E7 genes, in BCC-1/KMC cells. When E6 and E7 expressions were inhibited, IL-6/IL-8 expressions were repressed. Furthermore, IL-6/IL-8 remained inducible by UVB irradiation when E6 and E7 were inhibited. These results indicated that IL-6 and IL-8 can be upregulated by viral E6 and E7 proteins without UVB irradiation. Moreover, chronic exposure to UVB upregulates IL-6 and IL-8 when E6/E7 is induced by UVB.

Patchouli alcohol induces G0 /G1 cell cycle arrest and apoptosis in vincristine-resistant non-small cell lung cancer through ROS-mediated DNA damage.

BACKGROUND: Lung cancer, especially non-small cell lung cancer (NSCLC), is one of the leading causes of cancer-related deaths worldwide. Vincristine (VCR) is a chemotherapeutic agent for lung cancers; however, its effectiveness is limited by side effects and the development of drug resistance. Patchouli alcohol (PA), from Pogostemon cablin extract, is known to possess anti-inflammatory and anticancer properties. In this study, we investigated the role of PA in inducing reactive oxygen species (ROS)-mediated DNA damage in A549 and VCR-resistant A549/V16 NSCLC cells. METHODS: The anticancer potential of PA was studied using the MTT assay, colony formation, flow cytometry analysis, western blotting, DCFDA staining, immunofluorescence staining, and TUNEL assay techniques. RESULTS: The intracellular ROS levels were enhanced in PA-treated cells, activating the CHK1 and CHK2 signaling pathways. PA further inhibited proliferation and colony-forming abilities and induced cell cycle arrest at the G0 /G1 phase by regulating p53/p21 and CDK2/cyclin E1 expression. Moreover, PA increased the percentage of cells in the subG1 phase and induced apoptosis by activating the Bax/caspase-9/caspase-3 intrinsic pathway. In addition, drug resistance (p-glycoprotein) and cancer stem cell (CD44 and CD133) markers were downregulated after PA treatment. Furthermore, combining PA and cisplatin exhibited synergistic inhibitory activity in A549 and A549/V16 cells. CONCLUSIONS: PA induced ROS-mediated DNA damage, triggered cell cycle arrest and apoptosis, attenuated drug resistance and cancer stem cell phenotypes, and synergistically inhibited proliferation in combination with cisplatin. These findings suggest that PA has the potential to be used for the treatment of NSCLC with or without VCR resistance.

The alleviation effects of n-butylidenephthalide on apoptosis, senescence, and tight junction impairment of retinal pigment epithelium by activating Nrf-2/HO-1 signaling pathway in early diabetic retinopathy.

AIMS: Diabetic retinopathy (DR) is a common complication of diabetes that causes visual impairment and blindness in adults. This study aimed to explore the protective effects of n-Butylidenephthalide (BP) on hyperglycemia-treated RPE in vitro and in vivo. MAIN METHODS: C57BL/6 mice were injected with STZ by intraperitoneal to induce early DR and orally administrated with 2 mg/kg BP every day for twelve weeks. Body weight and blood glucose were measured once a week. The level of retina damage was determined by TUNEL assay and H&E staining. The outer blood-retinal barrier integrity and RPE65 expression of retina were evaluated by immunofluorescence. In in vitro study, ARPE-19 cells were long-term cultured with high glucose and BP for 8 days and studied for cell survival, tight junction integrity, RPE65 expression, angiogenic factors, mitochondria membrane potential (MMP), and ROS by MTT assay, Western blot, ß-galactosidase staining, immunofluorescence, JC-1, or DCFH-DA. KEY FINDINGS: The results indicate that BP suppressed the hyperglycemic effect and maintained retina anatomy normalization, as well as protected RPE cell survival, tight junction integrity, and RPE65 expression in vitro and in vivo. In vitro results showed BP stimulated high glucose-treated ARPE-19 cell proliferation and suppressed senescence via ERK pathway. Numerous ROS production and MMP imbalance were prevented by BP through Nrf-2/HO-1 pathway. BP inhibited high glucose-induced RPE neovascularization by VEGF dysregulation. SIGNIFICANCE: BP significantly protected tight junction integrity and RPE cellular physiology through ERK/Nrf-2/HO-1 pathway to prevent DR progression. Thus, BP has great potential to be developed therapeutic agents or adjuvants for DR.