Shikonin Inhibits Non-Small-Cell Lung Cancer H1299 Cell Growth through Survivin Signaling Pathway.

Overexpressed survivin is associated with worse survival of several types of human tumors. In this study, the antitumor activity of shikonin in non-small-cell lung cancer (NSCLC) by regulating survivin pathway was investigated. Results showed that shikonin inhibited the NSCLC H1299 cell proliferation in a dose-dependent manner. Moreover, shikonin fits well with survivin by molecular docking. Shikonin also inhibited the mRNA expression and protein level of survivin in H1299 cells. Shikonin arrested H1299 cell cycle at the G0/G1 phase by regulating CDK/cyclin family members. In addition, shikonin regulated the expression of X-linked inhibitor of apoptosis- (XIAP-) mediated caspases 3 and 9, thus leading to the damage of mitochondrial membrane potential and induction of H1299 cell apoptosis. Overall, shikonin inhibited H1299 cell growth by inducing apoptosis and blocking the cell cycle. The underlying mechanism involves targeting survivin, which subsequently regulates the protein expression of XIAP/caspase 3/9, CDK2/4, and cyclin E/D1. Thus, shikonin, a survivin inhibitor, is a promising therapeutic strategy in NSCLC treatment.

MicroRNA-26a systemic administration attenuates tumor formation in hepatocellular carcinoma mouse model.

MicroRNA (miRNA)-26a is one of the tumor suppressor genes that has been down regulated during the development of hepatocellular carcinoma (HCC). This work was conducted to evaluate the possible preventive effect of exogenous miRNA-26a administration on diethylnitrosamine (DEN)-mediated HCC. Balb/C mice were intraperitoneally injected with saline (Normal group), DEN (HCC group) or miRNA-26a (HCC+miRNA-26a group). On week 8, 12, 16 and 20, the concentrations of alpha-fetoprotein (AFP), des-gamma carboxyprothrombin (DCP), the levels of helper T cells-associated cytokines, and the vascular endothelial growth factor (VEGF), were measured. Flow cytometry determined the frequencies of regulatory T (Treg) cells. The concentrations of AFP, DCP and VEGF, as well as the frequency of Treg cells showed significantly lower values following miRNA-26a administration than in HCC group. miRNA-26a administration has reduced the levels of IL (interleukin)-2 and TNF (tumor necrosis factor)-α, in contrast, IL-10 level was markedly elevated in comparison to HCC model at all experimental time points. The restore of miRNA-26a function significantly (P<0.001) down regulated the expression levels of survivin & caspase-3 compared to HCC group. The obtained data introduce an evidence for the suppressive impact of miRNA-26a on liver tumor formation and its possible manipulation as a therapeutic design for HCC.

cRGD peptide-conjugated polyethylenimine-based lipid nanoparticle for intracellular delivery of siRNA in hepatocarcinoma therapy.

The effective delivery system plays an important role in the application of siRNA in the antitumor study. However, until now, researches on the delivery systems targeting hepatocarcinoma cells are still being explored. Here we designed and prepared a novel siRNA delivery system, cRGD-PSH-NP, which was based on a modified polyethyleneimine (PSH) and DSPE-PEG2000-cRGD. cRGD-PSH-NP loaded with survivin siRNA (cRGD-PSH-NP/S) was composed of egg phosphatidylcholine, cationic PSH, PEGylated lipids, survivin siRNA, and cRGD peptide as a targeting ligand. The formulations of cRGD-PSH-NP/S were optimized and characterized. In vitro investigations showed excellent gene silencing and antitumor activity compared with the unmodified nanoparticles in HepG2 cells. In vivo antitumor efficacy of cRGD-PSH-NP/S exhibited potent tumor inhibition (74.71%) in HepG2-bearing nude mice without inducing toxicity. These data suggested further research of cRGD-PSH-NP/S in hepatocarcinoma therapy.

Development of a carrier system containing hyaluronic acid and protamine for siRNA delivery in the treatment of melanoma.

The use of small interfering RNA (siRNA) in melanoma treatment remains limited owing to its biological properties. Herein, we developed a carrier system containing hyaluronic acid and protamine for siRNA delivery. Considering zeta potential and particle size as standards, the ratio of each component in liposome nanoparticles prepared was screened using the control variable method, and siRNA cationic liposome nanoparticles were prepared based on the optimal results obtained. The encapsulation rate of the cationic liposome nanoparticles was measured, and particle morphology was observed. B16F10 cells were treated with the nanoparticles; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, cell scratch experiments, and cell uptake experiments were performed to determine the effectiveness of the loaded siRNA. A mouse model was then established, and tumour tissues were subjected to haematoxylin-eosin staining. The inhibition of the survivin gene and protein expression were assessed using reverse transcription-polymerase chain reaction and western blotting, respectively. The results showed that the optimal mass ratio of hyaluronic acid (HA)-siRNA-to-protamine was 1.0; in the HA-siRNA-protamine complex containing 25 µg siRNA, the addition of 50 µL liposomes yielded optimal particles. And encapsulation rate was 85.07%. The nanoparticles demonstrated a significant inhibitory effect against melanoma cells; siRNA liposomes may inhibit tumour growth by down-regulating survivin. Survivin-siRNA cationic liposome nanoparticles could effectively inhibit the proliferation and migration of melanoma B16F10 cells in vitro and the proliferation of subcutaneous melanoma B16F10 cells, probably by inhibiting survivin mRNA and protein expression. Graphical abstract.

Specific expression of survivin, SOX9, and CD44 in renal tubules in adaptive and maladaptive repair processes after acute kidney injury in rats.

Acute kidney injury (AKI) is thought to be a reversible condition; however, growing evidence has suggested that AKI may be associated with subsequent development of chronic kidney disease. Although renal tubules have intrinsic regeneration capacity, disruption of the regeneration mechanisms leads to irreversible interstitial fibrosis. In this study, we investigated immunohistochemical markers of renal tubules in adaptive and maladaptive repair processes to predict AKI reversibility. Histopathological analysis demonstrated that regenerative tubules and dilated tubules were observed in the kidneys of AKI model rats after ischemia/reperfusion (I/R). Regenerative tubules gradually redifferentiated after I/R, whereas dilated tubules exhibited no tendency for redifferentiation. In fibrotic areas of the kidney in renal fibrosis model rats subjected to I/R, renal tubules were dilated or atrophied. There results suggested that the histopathological features of renal tubules in the maladaptive repair were dilation or atrophy. From microarray data of regenerative tubules, survivin, SOX9, and CD44 were extracted as candidate markers. Immunohistochemical analysis demonstrated that survivin and SOX9 were expressed in regenerative tubules, whereas SOX9 was also detected in renal tubules in fibrotic areas. These findings indicated that survivin and SOX9 contributed to renal tubular regeneration, whereas sustained SOX9 expression may be associated to fibrosis. CD44 was expressed in dilated tubules in the kidneys of AKI model rats and in the tubules of fibrotic areas of renal fibrosis model rats, suggesting that CD44 was expressed in renal tubules in maladaptive repair. Thus, these factors could be useful markers for detecting disruption of the regenerative mechanisms of renal tubules.

Myricanol 5-fluorobenzyloxy ether regulation of survivin pathway inhibits human lung adenocarcinoma A549 cells growth in vitro.

BACKGROUND: This study aimed to explore the growth inhibitory effect of myricanol 5-fluorobenzyloxy ether (5FEM) and its underlying mechanisms in human lung adenocarcinoma A549 cells in vitro. METHODS: 5FEM was obtained by the chemical modification of myricanol with fluorobenzyloxy ether at the OH(5) position. The cytotoxicity, cell apoptosis, cell cycle, mitochondrial membrane potential (ΔΨm), scratch test, colony formation, and the expression levels of the key survivin pathway-related genes in A549 were evaluated. RESULTS: 5FEM could significantly inhibit A549 cell growth; induce cell apoptosis; increase G0/G1 population; reduce ΔΨm; inhibit cell migration and colony formation; upregulate caspase-9, P21, and Bax expression levels; and downregulate PARP, survivin, and Bcl-2 expression level. CONCLUSION: These results enhanced our understanding of 5FEM and aid the discovery of novel myricanol derivatives as potential antitumor agents.

Grape Seed Proanthocyanidins Induce Autophagy and Modulate Survivin in HepG2 Cells and Inhibit Xenograft Tumor Growth in Vivo.

Liver cancer is one of the leading causes of death worldwide. Although radiotherapy and chemotherapy are effective in general, they present various side effects, significantly limiting the curative effect. Increasing evidence has shown that the dietary intake of phytochemicals plays an essential role in the chemoprevention or chemotherapy of tumors. In this work, HepG2 cells and nude mice with HepG2-derived xenografts were treated with grape seed proanthocyanidins (GSPs). The results showed that GSPs induced autophagy, and inhibition of autophagy increased apoptosis in HepG2 cells. In addition, GSPs also reduced the expression of survivin. Moreover, survivin was involved in GSPs-induced apoptosis. GSPs at 100 mg/kg and 200 mg/kg significantly inhibited the growth of HepG2 cells in nude mice without causing observable toxicity and autophagy, while inducing the phosphorylation of mitogen-activated protein kinase (MAPK) pathway-associated proteins, p-JNK, p-ERK and p-p38 MAPK and reducing the expression of survivin. These results suggested that GSPs might be promising phytochemicals against liver cancer.

Honokiol Enhances TRAIL-Mediated Apoptosis through STAMBPL1-Induced Survivin and c-FLIP Degradation.

Honokiol is a natural biphenolic compound extracted from traditional Chinese medicine Magnolia species, which have been known to display various biological effects including anti-cancer, anti-proliferative, anti-angiogenic, and anti-metastatic activities in cancer cells. Here, we found that honokiol sensitizes cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis through downregulation of anti-apoptotic proteins survivin and c-FLIP. Ectopic expression of survivin and c-FLIP markedly abolished honokiol and TRAIL-induced apoptosis. Mechanistically, honokiol induced protein degradation of c-FLIP and survivin through STAMBPL1, a deubiquitinase. STAMBPL1 interacted with survivin and c-FLIP, resulted in reduction of ubiquitination. Knockdown of STAMBPL1 reduced survivin and c-FLIP protein levels, while overexpression of STAMBPL1 inhibited honokinol-induced survivin and c-FLIP degradation. Our findings provided that honokiol could overcome TRAIL resistance through survivin and c-FLIP degradation induced by inhibition of STAMBPL1 expression.

Enhanced shRNA delivery by the combination of polyethylenimine, ultrasound, and nanobubbles in liver cancer.

BACKGROUND: Traditional cancer treatments such as surgery, radiation, and chemotherapy destroy both cancer and normal cells, which limit their clinical application. It is difficult to achieve the best results for any liver cancer patients using any single treatment method. Gene therapy for HCC demands non-invasive, efficient, targeted and safe gene transfection strategies. OBJECTIVE: In this study, a nonviral shRNA gene delivery system utilizing a combination of PEI, US, and NBs was developed for targeting survivin in liver Cancer. METHODS AND RESULTS: The PEI-shRNA-NBs cumulated in the tumor tissue because of the EPR effect. By exposure to the US, micelles shRNA may be released from PEI-shRNA-NBs in tumor tissues and the shRNA then transmitted efficiently to cancer cells. Considerably enhanced therapeutic outcome was obtained with the gene silencing effect enhanced. CONCLUSIONS: PEI-shRNA-NBs possess the potential to become promising tools intended for shRNA delivery.

Thymoquinone Enhances the Effect of Gamma Knife in B16-F10 Melanoma Through Inhibition of Phosphorylated STAT3.

BACKGROUND: Patients with brain metastasis from melanoma have a dismal prognosis with poor survival time. Gamma Knife (GK) is an effective treatment to control brain metastasis from melanoma. Thymoquinone (TQ) has emerged as a potential therapeutic option due to its antiproliferative effects on various cancers. The purpose of the study was to assess the effect of GK on B16-F10 melanoma cells in vitro and intracerebral melanoma in vivo, and its synergistic effect in combination with TQ. METHODS: The effects of GK and combination treatment of GK and TQ were studied on B16-F10 melanoma cells by evaluating cytotoxicity with an adenosine triphosphate assay, apoptosis by acridine orange staining, and genotoxicity by comet assay. Western blot analysis was performed to investigate the expression of STAT3, p-STAT3 (Tyr705), JAK2, p-JAK2, caspase-3, Bax, Bcl-2, survivin, and ß-actin. Expression of inflammatory cytokines was assessed by enzyme-linked immunosorbent assay. GK alone and in combination with TQ was assessed in an established intracerebral melanoma tumor in mice. RESULTS: The effects of GK on cytotoxicity, genotoxicity, and apoptosis were enhanced by TQ in B16-F10 melanoma cells. GK induced apoptosis through inhibition of p-STAT3 expression, which in turn regulated pro- and antiapoptotic proteins such as caspase-3, Bax, Bcl-2, and survivin. Adding TQ to GK irradiation further enhanced this apoptotic effect of GK irradiation. GK was shown to reduce the levels of tumor-related inflammatory cytokines in B16-F10 melanoma cells. This effect was more pronounced when TQ was added to GK irradiation. GK with 15 Gy increased the survival of mice with intracerebral melanoma compared with untreated mice. However, despite the additive effect of TQ in addition to GK irradiation on B16-F10 melanoma cells in vitro, TQ did not add any significant survival benefit to GK treatment in mice with intracerebral melanoma. CONCLUSIONS: Our findings suggest that TQ would be a potential therapeutic agent in addition to GK to enhance the antitumor effect of irradiation. Further studies are required to support our findings.

The anti-cancer effect of amygdalin on human cancer cell lines.

Derived from rosaceous plant seed, amygdalin belongs to aromatic cyanogenic glycoside group, and its anticancer effects have been supported by mounting evidence. In this study, we objected to investigate amygdalin effect on two antiapoptotic genes (Survivin, XIAP) and two lncRNAs (GAS5, MALAT1) in human cancer cells (A549, MCF7, AGS). Employing RT-qPCR analysis, we compared the mRNA levels of the genes related to apoptosis in A549, MCF7, and AGS cancer cells between amygdalin-treated (24, 48 and 72 h) and un-treated groups. RNA was extracted from both cell groups and then cDNAs were synthesized. The changes in the gene expression levels were specified using ΔΔCt method. RT-qPCR analysis has revealed that the expression of Survivin, XIAP, GAS5 and MALAT1 in amygdala-treated cancer cells were significantly different, compared to the un-treated cells. However, these expressions were different depending on the treatment time. According to the results, amygdalin significantly inhibited the expression level of Survivin, and XIAP genes in treated via untreated group. Our findings suggest that amygdalin might have an anticancer effect due to the various gene expressions in A549, MCF7, and AGS human cancer cells, showing it's potential as a natural therapeutic anticancer drug.

Anti-proliferative and anti-apoptotic potential effects of epigallocatechin-3-gallate and/or metformin on hepatocellular carcinoma cells: in vitro study.

The effects of epigallocatechin-3-gallate (EGCG) and metformin single treatment have been tested against hepatocellular carcinoma (HCC). This study aimed to assess the combination effects of EGCG and metformin on proliferation and apoptosis of HepG2cells and identified new potential molecular targets. The effect of EGCG and metformin against cell proliferation in HepG2 was determined using MTT assay. Reverse transcription polymerase chain reaction was applied to examine the gene expression of cyclin D1, lncRNA-AF085935, caspase-3, survivin and VEGF. The level of protein expression of glypican-3 was assessed by western blot. In HepG2 cells, EGCG and metformin combination treatment exhibited high significant effect against tumor proliferation. It significantly reduced cyclin D1, lncRNA-AF085935, glypican-3 and promoted apoptosis through increasing caspase3 and decreasing survivin compared to control cells. Moreover, EGCG and metformin treated cells showed decreased expression levels of VEGF. Our study provided new insights of the anticarcinogenic effects of EGCG and metformin on HCC through their effects on glypican-3 and lncRNA-AF085935.

Canine squamous cell carcinoma cell lines with high expression of survivin are sensitive to survivin inhibitor YM155.

Treatment of unresectable canine squamous cell carcinoma (SCC) remains challenging and new therapeutic strategies are needed. Survivin is a member of the inhibitor of apoptosis protein family and its inhibitor, YM155, is a potential anti-tumour agent. In the present study, 10 canine tumour cell lines (representing eight different tumour types) were screened for sensitivity to YM155; the drug potently inhibited the growth of the HAPPY SCC cell line. The growth inhibitory properties of YM155 were then examined in more detail using a panel of seven SCC cell lines. YM155 inhibited the growth of the cell lines HAPPY and SQ4; in contrast to the other lines in the panel, these two cell lines had high levels of expression of survivin. In HAPPY cells, YM155 inhibited expression of the survivin gene at the transcriptional level. In contrast, YM155 down-regulated survivin at the post-transcriptional level in SQ4 cells. YM155 suppressed cell growth in HAPPY cells, mostly via induction of apoptosis, but this was not the case in SQ4 cells. Two canine SCC cell lines with high cellular expression of survivin were sensitive to YM155. The possible underlying mechanisms of the cytotoxic effect of YM155 in these cell lines were different. One cell line had down-regulation of survivin mRNA and protein expression, associated with induction of apoptotic cell death. The other cell line had post-transcriptional down-regulation of survivin expression and subsequent induction of non-apoptotic cell death. Targeting survivin with YM155 is a potential approach for the treatment of canine SCCs with high expression of survivin.

Inhibitory effect of 11-carbonyl-beta-boswellic acid on non-small cell lung cancer H446 cells.

BACKGROUND: The anti-lung tumor potential of 11-carbonyl-ß-boswellic acid was investigated. MATERIALS & METHODS: The inhibitory effects of 11-carbonyl-ß-boswellic acid on non-small cell lung cancer (NSCLC) was assessed by proliferation, apoptosis, cell cycle and molecular mechanisms in NSCLC H446 cells in vitro. The results showed that the growth of H446 cells was significantly inhibited by 11-carbonyl-ß-boswellic acid in a dose- and time-dependent manner. Meanwhile, 11-carbonyl-ß-boswellic acid induced cell apoptosis and cell cycle G2-M phase arrest in H446 cells. RESULTS: Mechanistically, 11-carbonyl-ß-boswellic acid could activate JNK signaling pathway, down-regulate the expression of surviving protein, and activate the cleavage of PARP, leading to marked inhibitory effect on H446 cells. CONCLUSIONS: These findings suggest that 11-carbonyl-ß-boswellic acid may be a potential usefulness for preventing and treatment of NSCLC.

Both triazolyl ester of ketorolac (15K) and YM155 inhibit the embryonic angiogenesis in ovo (fertilized eggs) via their common PAK1-survivin/VEGF signaling pathway.

15 K is 1,2, 3-triazolyl ester of ketorolac, an old pain-killer, that blocks PAK1 by its R-form and inhibits COX-2 by its S-form. Mainly due to a robust increase in cell-permeability, 15K is over 500 times more potent than ketorolac in both anti-cancer and anti-PAK1 activities in cell culture with IC50 around 24 nM. However, 15K has no anti-AKT activity. Angiogenesis requires at least the kinase PAK1, and perhaps the kinase AKT as well, and is essential for a robust growth of solid tumors. Thus, in this study, we examined the potential antiangiogenic activity of 15K both in ovo and cell culture, prior to its in vivo (xenograft) anti-cancer activity test. The IC50 of 15K against the embryonic angiogenesis in ovo in CAM (chorioallantoic membrane) assay is around 1 nmol/egg. Surprizingly, however, 15K failed to inhibit the tube formation of HUVECs (human umbilical vein endothelial cells) in cell culture even at high as 150 µM. In an attempt to solve this mystery, we tested both in ovo as well as HUVECs-based anti-angiogenic activity of a potent survivin-suppressor called YM155, which blocks PAK1, in addition to AKT. YM155 is slightly more potent than 15K in CAM assay with IC50 around 0.5 nmol/egg, and apparenty inhibits the tube formation of HUVECs with IC50 around 18 nM. According to a few previous findings with the direct PAK1-inhibitor frondoside A (FRA), the tube formation of HUVECs depends solely on PAK1. Thus, the failure of 15K to affect their tube formation is most likely due to their drug (15K)-resistance. Furthermore, unlike FRA, YM155 killed HUVECs with IC50 around 18 nM, clearly indicating that AKT is essential for survival of HUVECs, instead of their tube formation.