Metformin reverses PARP inhibitors-induced epithelial-mesenchymal transition and PD-L1 upregulation in triple-negative breast cancer.

Poly (ADP-ribose) polymerase (PARP) inhibitors have emerged as promising targeted therapies for BRCA-mutated cancers by blocking repair of DNA double-strand breaks. However, resistance to PARP inhibitors (PARPi) has been described in some patients lowering the overall response rates. To investigate the underlying mechanisms of PARPi resistance, we developed the adaptive resistant clones in triple-negative breast cancer cell lines. We identified epithelial-mesenchymal transition (EMT) and upregulation of programmed death-ligand 1 (PD-L1) in resistant cells and further demonstrated the important role of Akt S473 phosphorylation in PARPi resistance. In addition, PARPi mediated EMT is independent of PD-L1 upregulation. Blocking the p-Akt S473 axis by metformin reversed EMT and PD-L1 expression which sensitized PARPi-resistant cells to cytotoxic T cells. Thus, a combination of metformin and PARP inhibitors may be a promising therapeutic strategy to increase the efficacy of PARP inhibitors and tumor sensitivity to immunotherapy.

Synergism of PARP inhibitor fluzoparib (HS10160) and MET inhibitor HS10241 in breast and ovarian cancer cells.

Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) are promising targeted therapeutics for breast and ovarian cancers bearing a germline BRCA1/2 mutation (BRCA m), and several have already received regulatory approval in the United States. In patients with a BRCA m cancer, PARPi can increase the burden of unrepaired DNA double-strand breaks by blocking PARP activity and trapping PARP1 onto damaged DNA. Resistance to PARP inhibitors can block the formation of DNA double-strand breaks through BRCA-related DNA repair pathway. MET is a hyper-activated receptor tyrosine kinase expressed in multiple cancer types and the activation contributes to resistance to DNA damage-inducing therapeutic drugs. Our previous study showed that MET inhibition by pan-kinase inhibitors has synergism with PARPi in suppressing growth of breast cancer in vitro and in xenograft tumor models. In this study, we validated the inhibitory effect of novel inhibitors, HS10241 (selective MET inhibitor) and HS10160 (PARPi), to their target respectively in triple-negative breast cancer (TNBC) and high-grade serous ovarian cancer (HGSOC) cells. We further demonstrated that these two inhibitors function synergistically in eliminating TNBC and HGSOC cells; combining with HS10241 increased DNA double-strand breaks induced by HS10160 in cancer cells; and PARP1 tyrosine (Y)-907 phosphorylation (PARP1 p-Y907) can be an effective biomarker as an indicator of MET-mediated PARPi in HGSOC. Our results suggest that the combination of HS10241 and HS10160 may benefit patients bearing tumors overexpressing MET as well as those resistant to single-agent PARPi treatment.

Preparation of polyphosphazene hydrogels for enzyme immobilization.

We report on the synthesis and application of a new hydrogel based on a methacrylate substituted polyphosphazene. Through ring-opening polymerization and nucleophilic substitution, poly[bis(methacrylate)phosphazene] (PBMAP) was successfully synthesized from hexachlorocyclotriphosphazene. By adding PBMAP to methacrylic acid solution and then treating with UV light, we could obtain a cross-linked polyphosphazene network, which showed an ultra-high absorbency for distilled water. Lipase from Candida rugosa was used as the model lipase for entrapment immobilization in the hydrogel. The influence of methacrylic acid concentration on immobilization efficiency was studied. Results showed that enzyme loading reached a maximum of 24.02 mg/g with an activity retention of 67.25% when the methacrylic acid concentration was 20% (w/w).

Induction of biliary cholangiocarcinoma cell apoptosis by 103Pd cholangial radioactive stent gamma-rays.

BACKGROUND: In recent years, interventional tumor therapy, involving implantation of intra-cholangial metal stents through percutaneous trans-hepatic punctures, has provided a new method for treating cholangiocarcinoma. (103)Pd cholangial radioactive stents can concentrate high radioactive dosages into the malignant tumors and kill tumor cells effectively, in order to prevent re-stenosis of the lumen caused by a relapsed tumor. The aim of the present study was to investigate the efficacy of gamma-rays released by the (103)Pd biliary duct radioactive stent in treating cholangiocarcinoma via induction of biliary cholangiocarcinoma cell apoptosis. METHODS: A group of biliary duct cancer cells was collectively treated with a dose of gamma-rays. Cells were then examined by the 3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl terazolium-bromide (MTT) technique for determining the inhibition rate of the biliary duct cancer cells, as well as with other methods including electron microscopy, DNA agarose gel electrophoresis, and flow cytometry were applied for the evaluation of their morphological and biochemical characteristics. The growth curve and the growth inhibition rate of the cells were determined, and the changes in the ultrastructure of the cholangiocarcinoma cells and the DNA electrophoresis bands were examined under a UV-lamp. RESULTS: The gamma-ray released by (103)Pd inhibited cholangiocarcinoma cell growth, as demonstrated when the growth rate of the cells was stunned by a gamma-ray with a dosage larger than 197.321 MBq. Typical features of cholangiocarcinoma cell apoptosis were observed in the 197.321 MBq dosage group, while cell necrosis was observed when irradiated by a dosage above 245.865 MBq. DNA agarose gel electrophoresis results were different between the 197.321 MBq irradiation dosage group, the 245.865 MBq irradiation dosage group, and the control group. CONCLUSIONS: (103)Pd radioactive stents which provide a radioactive dosage of 197.321 MBq are effective in the treatment of cholangiocarcinoma; (103)Pd radioactive stents should be useful for the clinical treatment of cholangiocarcinoma.

103Pd-induced apoptosis of proliferative smooth muscle cells in bile ducts of dogs: significance and effects on related genes.

BACKGROUND: With the objective of developing a locally-produced radioactive stent, the present study used in vivo animal experiments to explore apoptosis of proliferative smooth muscle cells resulting from facilitation of the expression of genes caused by gamma-radiation in order to prevent bile duct restenosis. We therefore explored the effects and significance of gamma-radiation on the activity of caspase-3, Fas and Bcl-2 genes in apoptosis of proliferative smooth muscle cells in the bile duct walls of dogs. METHODS: Twelve dogs were randomly divided into 2 groups (6 in each group). A postinjury bile duct stenosis model was established and radioactive (103)Pd ((103)palladium) or ordinary bile duct stents were implanted into the bile ducts. HE staining, RT-PCR and immunohistochemistry were used to detect the proliferation and apoptosis of bile duct smooth muscle cells in proliferative endomembrane and the expression of related caspase-3, Bcl-2 and Fas genes. RESULTS: The expression of caspase-3 and Fas genes in the bile duct tissues of dogs with radioactive stents was higher than that of dogs with ordinary stents. There was significant apoptosis of proliferative smooth muscle cells in the bile ducts. The expression of the Bcl-2 gene in the bile duct tissues of dogs with radioactive stents was lower than that in those with ordinary stents. There was significant apoptosis of proliferative smooth muscle cells in the dogs with low Bcl-2 gene expression. CONCLUSIONS: Radiation increases the activity of caspase-3 and Fas genes and is associated with apoptosis. The radioactive (103)Pd stent may facilitate apoptosis of proliferative smooth muscle cells in the bile ducts of dogs by activating these genes. The Bcl-2 gene expression level is correlated with the occurrence of apoptosis and the radiosusceptibility of cells.

103Pd radioactive stent inhibits biliary duct restenosis and reduces smooth muscle actin expression during duct healing in dogs.

BACKGROUND: This study was designed to assess the expression of smooth muscle actin (SMA) in the healing process after implanting a (103)Pd radioactive stent in the biliary duct, and to discuss the function and significance of this stent in preventing biliary stricture formation. METHODS: A model of biliary injury in dogs was made and then a (103)Pd radioactive stent was positioned in the biliary duct. The expression and distribution of SMA were assessed in the anastomotic tissue 30 days after implantation of the stent. RESULTS: SMA expression was less in the (103)Pd stent group than in the ordinary stent group. The (103)Pd stent inhibited scar contracture and anastomotic stenosis. CONCLUSION: The (103)Pd stent can reduce the expression of SMA in the healing process and inhibit scar contracture and anastomotic stenosis in the dog biliary duct.

[Expression and significance of caspase-3 gene in apoptotic muscle cells 103Pd radioactive stent bile duct in dogs].

OBJECTIVE: To discuss the expression and significance of caspase-3 gene in the apoptotic muscle cells in gamma-radiation-induced muscle cell lines. METHODS: The caspase-3 mRNA in the control and gamma-radiation induced apoptotic muscle cells was analysed by RT-PCR. RESULTS: The expression of caspase-3 gene transcript was higher in 103Pd radioactive stent dog bile duct than in general stent dog bile duct, and apoptotic muscle cells were higher in 103Pd radioactive stent dog bile duct than in general stent dog bile duct. CONCLUSIONS: The high level expression of caspase-3 gene may help to understand the muscle cells sensitivity to gamma-radiation apoptosis. 103Pd radioactive stent may increase the expression of caspase-3 gene in dog bile duct and prevent the billiary narrow when dog bile duct was injured by balloon.

[Effect of 103palladium radioactive stent on expression of smooth muscle actin in bile duct during healing process and its significance].

OBJECTIVE: To observe the effect of radiation on the expression of smooth muscle actin (SMA) in the bile duct during the healing process and the inhibitory function of (103)palladium (Pd) radioactive stent on the stricture of bile duct after injury. METHODS: Twelve mongrel dogs were made models of bile duct injury: duodenotomy was performed, a balloon catheter was inserted into the general bile duct and saline with high pressure was perfused thereinto to cause laceration of the mucosa, and then the balloon catheter was withdrawn and ordinary alloy stent or (103)Pd radioactive stent was inserted into the general bile duct. Thirty days after the dogs were killed. Their bile ducts were taken out to undergo HE staining to observe the area of general bile duct, thickness of the tunica intima, area of residual bile duct cavity, stricture degree, and circumference of bile duct. The expression of SMA in the bile duct tissue was detected by immunoistochemistry. RESULTS: SMA was expressed in 5 of the 6 specimens of bile duct in the (103)Pd radioactive stent group and 2 of the 6 specimens of the ordinary stent group (P < 0.01). The maximum thickness of tunica intima of general bile duct was 0.78 mm +/- 0.12 mm in the (103)Pd radioactive stent group, significantly less than that of the ordinary stent group (1.86 mm +/- 0.14 mm, P < 0.01). The percentage of maximum stricture area of the (103)Pd radioactive stent group was 23% +/- 16%, significantly lower that that of the ordinary stent group (56% +/- 22%, P < 0.01). The circumference of bile duct cavity of the (103)Pd radioactive stent group was 9.7 mm +/- 1.6 mm, significantly longer that of the ordinary stent group (7.0 mm +/- 1.4 mm, P < 0.01). CONCLUSION: (103)Pd radioactive stent reduces the expression of SMA in the bile duct during the healing process, thus inhibiting the stricture of bile duct caused by scar contracture at the anastomotic stoma.

Intrabiliary radiation inhibits smooth muscle formation and biliary duct remodelling after balloon overstretching injury in dogs.

BACKGROUND: Internal metallic stents have been widely used in clinical practice, but a high postoperative restenosis rate limits its application. The purpose of this study was to determine the effect of intrabiliary radiation on muscle formation and biliary duct remodeling after biliary duct balloon injury in dogs. METHODS: Twenty male dogs (15 - 20 kg) were randomly divided into treatment group (n = 10) and control group (n = 10). Balloon overstretching injury was induced using a balloon catheter placed across the biliary duct. Subsequently, a 103Pd radioactive stent was positioned at the target site in each animal in the treatment group, providing the injured biliary duct with a radiation dose of 12.58 x 10(7) Bq. Dogs in the control group received Ni-Ti stents. All the dogs were killed one month after initial injury. The injured sections were dissected free from the dogs, and were processed for histological and morphological study. Cross-sections were stained with hematoxylin-eosin, Masson's trichrome, and Verhoef-van Giesen. Muscle formation area and lumen area were determined using a computer-assisted image analysis system. RESULTS: Compared with the control group, 103Pd radioactive stents significantly reduced muscle formation area (78.3%, P < 0.01), and percentage area of stenosis [control stents: (60.0 +/- 21.6)%, 103Pd radioactive stents: (31.6 +/- 9.5)%]. In addition, in the treatment group, the biliary duct lumen area was significantly larger than that in the control group (P < 0.01). CONCLUSIONS: 103Pd radioactive stents providing a radioactive dose of 12.58 x 10(7) Bq are effective in reducing muscle formation and biliary duct remodeling after balloon overstretching injury.