The Impact of Terminal Peptide Extensions of Retinal Inosine 5´Monophosphate Dehydrogenase 1 Isoforms on their DNA-binding Activities.

The main structural difference between the mutation-susceptible retinal isoforms of inosine 5´-monophosphate dehydrogenase-1 (IMPDH-1) with the canonical form resides in the C- and N-terminal peptide extensions with unknown structural/functional impacts. In this report, we aimed to experimentally evaluate the functional impact of these extensions on the specific/non-specific single-stranded DNA (ssDNA)-binding activities relative to those of the canonical form. Our in silico findings indicated the possible contribution of the C-terminal segment to the reduced flexibility of the Bateman domain of the enzyme. In addition, the in silico data indicated that the N-terminal tail acts by altering the distance between the tetramers in the concave octamer complex (the native form) of the enzyme. The overall impact of these predicted structural variations became evident, first, through higher Km values with respect to either of the substrates relative to the canonical isoform, as reported previously (Andashti et al. in Mol Cell Biochem 465(1):155-164, 2020). Secondary, the binding of the recombinant mouse retinal isoform IMPDH1 (603) to its specific Rhodopsin target gene was significantly augmented while its binding to non-specific ssDNA was lower than that of the canonical isoform. The DNA-binding activity of the other mouse retinal isoform, IMPDH1(546), to specific and non-specific ssDNA was lower than that of the canonical form most probably due to the in silico predicted rigidity created in the Bateman domain by the C-terminal peptide extension. Furthermore, the DNA binding to the Rhodopsin target gene by each of the IMPDH isoforms influenced in the presence of GTP (Guanosine triphosphate) and ATP (Adenosine triphosphate).

Biochemical and histopathological evaluation of ChAdOx1-S and BBIBP-CorV vaccination in normal and streptozotocin-induced diabetic Wistar rats.

Infection with Covid-19 has been associated with some medical complications namely diabetes, thrombosis, and hepatic and renal dysfunction among others. This situation has created some concern about the use of relevant vaccines which might cause similar complications. In that regard, we planned to evaluate the impact of two of the relevant vaccines namely ChAdOx1-S and BBIBP-CorV on some of the blood biochemical factors and also on liver and kidneys functions following the immunization of healthy and streptozotocin-induced diabetic rats. Evaluation of the level of neutralizing antibody among the rats indicated that immunization with ChAdOx1-S induced a higher level of neutralizing antibody among both the healthy and diabetic rats compared to the BBIBP-CorV vaccine. Furthermore, the neutralizing antibody levels against both types of vaccines were significantly lower in diabetic rats than in healthy ones. On the other hand, no alterations were observed in the rats' sera biochemical factors, coagulation values and histopathological images of the liver and kidneys. Altogether these data besides of confirming the effectiveness of both vaccines, indicate that both vaccines have no hazardous side effects on rats and probably humans though clinical investigations are required to validate our present data.

Beta cell regeneration upon magainin and growth hormone treatment as a possible alternative to insulin therapy.

Insulin therapy, pancreas transplantation and ß cell regeneration are among the suggested treatment strategies for type 1 diabetes. It has been shown that some antimicrobial peptides have the potential to increase insulin release and to improve glucose tolerance, although the mechanism by which they promote the regeneration of damaged pancreatic cells to functional ß-like cells remains unknown. To answer this question, we evaluated the in vivo effects of magainin-AM2 and growth hormone (GH) on the regeneration of streptozotocin (STZ)-damaged mouse pancreas. Treatment with magainin-AM2 and GH ameliorated the effects of STZ on fasting blood glucose and glucose tolerance test values, and also resulted in a significant increase in total cell counts (α and ß) and the number of insulin+ and glucagon+ cells per islet and a decrease in the number of T and B cells. In addition, we observed a 1.43- and 2.21-fold increase in expression of paired box 4, one of the main factors for α to ß-like cell conversion, in normal- and diabetes-treated mice, respectively. Similarly, expression of P-S6 and extracellular signal-regulated kinases 1 and 2, required for cell proliferation/differentiation, increased by 3.27- and 2.19-fold among the diabetes-treated and control diabetic mice, respectively. Furthermore, in all experiments, amelioration of the effects of STZ were greatest upon Mag treatment followed by GH administration. The present in vivo data provide evidence in support of the possibility of pharmaceutical induction of α cell production and their trans-differentiation to functional ß-like cells.

Teucrium polium L: An updated review of phytochemicals and biological activities.

Objective: Medicinal plants and their components are potential novel sources for developing drugs against various diseases. Teucrium polium L. (syn Teucrium capitatum L. or felty germander) from the Lamiaceae family, is widely distributed in the dry and stony places of the hills and deserts of almost all Mediterranean countries, southwestern Asia, Europe, and North Africa. Based on traditional Iranian medicine (TIM), T. polium is used for treating many diseases, including abdominal pain, indigestion, and type 2 diabetes. Materials and Methods: In our previous review article published in 2012 and based on 100 articles published from 1970 to 2010, the main compounds purified from T. polium were terpenes, terpenoids, and flavonoids with antioxidant, anticancer, anti-inflammatory, hypoglycemic, hepatoprotective, hypolipidemic, antibacterial, and antifungal activities. Results: In this article, the phytochemistry and pharmacological activities of the plant reported from 2011 to 2020 have been evaluated. Therefore, a search was done in the databases PubMed, Science Direct and Google Scholar, Scopus, and Web of Science with the terms "T. polium," "T. capitatum." and felty germander', which included about 100 articles published since 2011 about T. polium pharmacological activities and isolated compounds. Most studies of this review focused on the antioxidant and antidiabetic effects of the plant. Conclusion: Considering the position of T. polium in folk medicine, mainly as an antidiabetic agent, purification, structural and biological characterization of the active components appears essential for effective use of the plant.

Differentiation of PANC-1 ductal cells to ß-like cells via cellular GABA modulation by Magainin and CPF-7 peptides.

Some of the antimicrobial peptides induce insulin release and improve glucose tolerance while their effects on pancreatic cell differentiation have remained unresolved. In this report, we evaluated the effects of two of these peptides, Magainin-II and CPF-7, and also GABA, on PANC-1 ductal cells' differentiation. Based on immunofluorescence and qRT-PCR analyses the expression levels of some of the Epithelial to Mesenchymal transition (EMT)-related factors such as Snai1 and Ngn3, as two biomarkers of alpha and beta cells, were increased. Our findings also revealed a drastic increase in Arx, Pax4, Dnmt-1 and Glucagon expressions associated with dedifferentiation of PANC-1 cells into pancreatic endocrine progenitor cells. Futhermore, Magainin-II and CPF-7 exerted their roles partly via influencing the GABA cellular content. These data would undoubtedly provide a suitable ground for further investigation to guide these cells toward transplantable insulin producing beta cells.

Terminal Peptide Extensions Augment the Retinal IMPDH1 Catalytic Activity and Attenuate the ATP-induced Fibrillation Events.

Defects in inosine monophosphate dehydrogenase-1 (IMPDH1) lead to insufficient biosyntheses of purine nucleotides. In eyes, these defects are believed to cause retinitis pigmentosa (RP). Major retinal isoforms of IMPDH1 are structurally distinct from those in other tissues, by bearing terminal extensions. Using recombinant mouse IMPDH1 (mH1), we evaluated the kinetics and oligomerization states of the retinal isoforms. Moreover, we adopted molecular simulation tools to study the possible effect of terminal tails on the function of major enzyme isoforms with the aim to find structural evidence in favor of contradictory observations on retinal IMPDH1 function. Our findings indicated higher catalytic activity for the major mouse retinal isoform (mH1603) along with lower fibrillation capacity under the influence of ATP. However, higher mass oligomerization products were formed by the mH1 (603) isoform in the presence of the enzyme inhibitors such as GTP and/or MPA. Collectively, our findings demonstrate that the structural differences between the retinal isoforms have led to functional variations possibly to justify the retinal cells' requirements.

Investigation of 3T3-L1 Cell Differentiation to Adipocyte, Affected by Aqueous Seed Extract of Phoenix Dactylifera L.

BACKGROUND: Obesity, often associated with insulin resistance and type 2 diabetes, is a metabolic disease that can result in dyslipidemia and hyperglycemia. Many reports describe the hypoglycemic and hypolipidemic properties of the Phoenix dactylifera L. seed extract in STZ-induced diabetic rat models, however, its anti-diabetic effects in other diabetic models are less characterized in the literature. This study set out to determine the possible effects of the Phoenix dactylifera L. seed extract on adipogenesis and glucose homeostasis. METHODS: 3T3-L1 cells were cultured in adipocyte differentiation media with or without varying doses of Phoenix dactylifera L. extract (0.312-1 mg/ml). Assays were performed on days 5, 8, and 12 after induced differentiation. RESULTS: Our results demonstrate that the triglyceride content in treated groups was significantly lower compared to controls. Further, treating 3T3-L1 cells with Phoenix dactylifera L. seed extract reduced adipogenesis through the downregulation of PPAR-γ and CEBP-α, and adipocyte-specific genes involved in fatty acid metabolism including ap2, ACACA, and FAS. CONCLUSION: Phoenix dactylifera L. seeds have the potential to inhibit adipogenesis and obesity. Overall, this study explored the inhibitory effects of Phoenix dactylifera L. seed extract on adipogenesis in 3T3-L1 cells on the molecular level.

The functional impact of the C/N-terminal extensions of the mouse retinal IMPDH1 isoforms: a kinetic evaluation.

Mutations in the retinal inosine monophosphate dehydrogenase1 (IMPDH1) gene is believed to be one cause of retinitis pigmentosa (RP). The main structural difference between the mutation-susceptible retinal isoforms with canonical one resides in the C- and N-terminal extensions. There are limited studies on the structure and function of terminal peptide extensions of the IMPDH1 retinal isoforms. Using recombinant murine IMPDH1 (mH1), we evaluated the kinetics of the retinal isoforms along with inhibition by some of the purine nucleotides. Molecular modeling tools were also applied to study the probable effect(s) of the terminal peptide tails on the function of the retinal isoforms. Molecular dynamic simulations indicated the possible impact of the end-terminal segments on the enzyme function through interactions with the enzyme's finger domain, affecting its critical pseudo barrel structure. The higher experimentally-determined Km and Ki values of the retinal mIMPDH1 (546) and mIMPDH1 (603) relative to that of the canonical isoform, mIMPDH1 (514), might clearly be due to these interactions. Furthermore and despite of the canonical isoform, the retinal isoforms of mH1 exhibited no NAD+ substrate inhibition. The resent data would certainly provide the ground for future evaluation of the physiological significance of these variations.

A genome-scale CRISPR/Cas9 knockout screening reveals SH3D21 as a sensitizer for gemcitabine.

Gemcitabine, 2',2'-difluoro-2'-deoxycytidine, is used as a pro-drug in treatment of variety of solid tumour cancers including pancreatic cancer. After intake, gemcitabine is transferred to the cells by the membrane nucleoside transporter proteins. Once inside the cells, it is converted to gemcitabine triphosphate followed by incorporation into DNA chains where it causes inhibition of DNA replication and thereby cell cycle arrest and apoptosis. Currently gemcitabine is the standard drug for treatment of pancreatic cancer and despite its widespread use its effect is moderate. In this study, we performed a genome-scale CRISPR/Cas9 knockout screening on pancreatic cancer cell line Panc1 to explore the genes that are important for gemcitabine efficacy. We found SH3D21 as a novel gemcitabine sensitizer implying it may act as a therapeutic target for improvement of gemcitabine efficacy in treatment of pancreatic cancer.

Overexpression of Hes1 is involved in sensitization of K562 cells to Imatinib.

Tyrosine kinase inhibitor (TKI)-based therapy has created promising results among much chronic myeloid leukemia (CML) patients. Imatinib as a relatively specific inhibitor of Bcr-Abl is at present one of the undisputed therapeutic agent for newlydiagnosed patients with CML. However, the occurrence of imatinib-resistance enlightens the urgent need to identify other therapeutic agents against CML. Juglone (5-hydroxy-2-methyl-1, 4-naphthoquinone) exerts cytotoxic effects against various human cancer cell lines. However, the mechanisms through which Juglone induces anticancer effects in CML especially in comparison with imatinib treatment remain unknown. Our results revealed that Juglone-inhibited K562 cells growth through inducing apoptosis. Based on our Western blot analyses, Juglone significantly reduced p-Akt levels and increased the expression level of Forkhead box O1 (FoxO1) and FoxO3a proteins. Moreover, hairy/enhancer of split-1 (Hes1) protein, overexpressed under the influence of Juglone, is apparently involved in Juglone-induced apoptosis among K562 cells. Conversely, treatment with imatinib attenuated Hes1 protein expression. Considering the different functional mechanism of Juglone compared with imatinib, it seems that Juglone treatment could be a useful alternative strategy for the treatment of patients with imatinib-resistance.

Hypolipidemic activity of Dracocephalum kotschyi involves FOXO1 mediated modulation of PPARγ expression in adipocytes.

BACKGROUND: Dracocephalum kotschyi, as a wild-growing flowering plant (from Lamiaceae family), is locally prescribed for its various health-promoting properties including hypolipidemic and hypoglycemic effects. To evaluate the scientific basis of the traditional use of Dracocephalum kotschyi extract (DKE), we aimed to disclose its mode of action with main focus on white adipose tissue of diabetic rats. METHODS: Streptozotocin-induced diabetic rats were exposed to different doses of DKE for 28 days followed by the determination of the sera biochemical factors. The oxidative stress status of the diabetic versus nondiabetic rats' adipose tissue under the influence of DKE were also evaluated in terms of malondialdehyde (MDA) and some of antioxidant enzymes (superoxide dismutase, SOD, and catalase). Furthermore, we exposed 3T3-L1 cells to DKE and then evaluated both the extent of cells differentiation to adipocytes and measured the expression levels of some of the key signaling elements involved in adipogenesis and lipogenesis with main focus on PPARγ. RESULTS: Our results indicated that DKE administration attenuated the levels of TG (triglycerides), TC (total cholesterol), LDL and blood glucose by 54, 40, 54 and 25%, respectively and enhanced the levels of HDL, catalase and SOD by 45, 74 and 56%, respectively. In addition to profound adipogenic and lipogenic effects on 3T3-L1 cells, DKE significantly enhanced p-AKT, p-FOXO1, PPARγ and SREBP-1 expressions while that of p-JNK was quenched parallel to effect of pioglitazone, an antidiabetic agent, used in our investigation as the positive control drug. CONCLUSIONS: Besides of confirming the hypolipidemic action of the plant, our results provided documents on at least one mode of action of DKE with profound effect on lipid metabolism in adipose tissue. Regarding our results, further investigation on DKE, as a new potential hypolipidemic alternative drug is warranted.

Augmentation of endogenous GABA pool size induced by Magainin II peptide.

BACKGROUND: Gamma aminobutyric acid (GABA), an inhibitory neurotransmitter, is produced via decarboxylation of l-glutamate through the glutamic acid decarboxylase (GAD) enzyme. The synchronic action of GABA-transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH) enzymes convert the GABA metabolite into succinate. Given this background, our research was aimed at probing the effect of Magainin II, on the activity of GABA shunt metabolizing enzymes. METHODS: Male NIH mice were administered peripherally by Magainin II (50 µg/kg body weight) and saline solution (%0.9 (w/v)) as the control vehicle. At different time intervals, the mice were sacrificed to evaluate the effect of Magainin II injection on the GABA shunt pathway. The activity of hypothalamic GAD, GABA-T and SSADH enzymes were determined using relevant enzyme assays. RESULTS: Magainin II effectively enhanced the activity of GAD, by %90, 24 h after injection, while quenching the activities of GABA-T and SSADH by %43 and %71, respectively. In vitro models also revealed the direct but reversible interaction between the peptide and each of the individual enzymes of GABA shunt pathway. CONCLUSION: This study confirms the probable role of Magainin II in increasing the GABA content of the mouse hypothalamus. This property might candidate the peptide as a novel agent for improving the symptoms of many GABA dependent psychiatric disorders.

Induction of transient cell cycle arrest by H2 O2 via modulation of ultradian oscillations of Hes1, Socs3, and p-Stat3 in fibroblast cells.

Biological clocks, time-keeping systems, enable the living organisms to synchronize their biochemical processes with their environment. Among these molecular oscillators, ultradian oscillators have been identified with volatility less than 24 h. Transcription factor Hes1, a member of the basic Helix-loop-Helix (bHLH) protein family, has an oscillation duration of 2 h in vertebrates. Due to the pivotal role of oxidative stress in many human diseases, we evaluated the effect(s) of oxidative stress on Hes1 oscillator, its upstream regulators, and its downstream cell cycle regulators. NIH/3T3 mouse fibroblast cells were treated with sublethal (250 µM) and lethal (1000 µM) doses of H2 O2 for 30 min. H2 O2 generated a delay in p-Stat3 and Socs3 mRNAs followed by suppression of Hes1 protein. These events were accompanied by simultaneous upregulation of p21 and downregulation of cyclinD1, resulting in a temporary arrest of the cell cycle. In conclusion, the elimination of Hes1 protein oscillation by H2 O2 may represent a defense mechanism against oxidative stress in fibroblast cells.

A simplified globally affordable experimental setup for monitoring DNA diagnosis by a QD-based technique.

The unavailability of simple, quick, and sensitive genetic-based molecular diagnostic techniques has become the main driving force for inventing new approaches in the era of quantum dots (QDs): a new class of fluorescent probes with fascinating optical electronic properties. Using the unique size-dependent light-emitting properties of QDs, we have developed a QD-based ultrasensitive technique which removes the necessity for the genetic amplification step required in almost all types of molecular-based diagnostic techniques. The selectivity of the new approach is warranted by the careful design of a pair of specific oligonucleotide probes, chemically modified at their 5'-ends. Our results indicated the selective detection of Salmonella typhi in an assay time of 50 min with a limit of detection (LOD) of 2 CFU/mL. The rapidity, selectivity, and sensitivity and the low assay cost make the new diagnostic technique a promising new tool for laboratory and field-based approaches to molecular diagnosis of health-threatening pathogens. Graphical abstract.

Differential impact of various reactive oxygen species (ROS) on HIF-1α/p53 direct interaction in SK-N-MC neuroblastoma cells.

BACKGROUND: A vital property of eukaryotic cells physiology is their rather quick response to variation of oxygen tension, mainly by a transcription factor known as hypoxia-inducible factor-1 (HIF-1). Aside from its transcriptional regulation, other mechanisms, such as post translational modifications and protein-protein interactions, the interaction between HIF-1α and p53 has attracted more attention mainly due to simultaneous enhancement in the protein levels of these two anti- and pro-apoptotic vital transcriptional factors within the ROS-stressed cells. METHODS: In this study, we measured cell viability following exposure of the cells to H2O2, menadione and Cobalt Chloride by MTT, and ROS content was measured under the same condition. The immunoblotting technique has been used to establish the presence and amount of Caspase, HIF-1α and p53 proteins. Then, the effect of different ROS on interaction between HIF-1α and p53 proteins was examined by co-immunoprecipitation. RESULTS: The results showed that cells viability and intracellular ROS content were modulated in response to menadione, H2O2 and Cobalt Chloride. These agents had different influence on HIF-1α signaling pathways as well as its interactions with p53 protein. It appeared that direct communication between HIF-1α and p53 proteins by ROS stresses, under both normoxic and hypoxic conditions, was governed by HIF-1α at a certain induced level. CONCLUSIONS: Our data indicated that stabilization, a prerequisite for communication, of HIF-1α is dependent to the types of free radicals.

Teucrium polium extract reverses symptoms of streptozotocin-induced diabetes in rats via rebalancing the Pdx1 and FoxO1 expressions.

BACKGROUND AND OBJECTIVE: Teucrium polium Lamiaceae. an antidiabetic traditional medicine was observed to stimulate insulin secretion and ß-cell mass restoration in pancreatic of diabetic rats. Bioactive compounds in T. polium such as terpenoids and flavonoids have a wide range of antioxidant properties. Given this background, our research was aimed at probing the effective molecular mechanism of T. polium which in turn protects the pancreatic ß-cells of diabetic rats through attenuation of streptozotocin-induced oxidative stress. MATERIALS AND METHODS: Induction of diabetes by streptozotocin (STZ) injection followed by treatment of the rats with T. polium extract (0.5g/kg) and glibenclamide as a standard drug (600µg/kg) for six consecutive weeks. Besides, biochemical indexes including sera glucose and lipids were investigated once every two weeks. Moreover, oxidative stress markers were also measured in the sixth week of treatment to assess the antioxidant capacity of T. polium. Prior to sacrifice of rats, the oral glucose tolerance test was carried out and thereafter western blot analysis was performed on pancreatic tissues to detect the expression of JNK, FoxO1 and Pdx1 proteins in the experimental groups. RESULTS: Oral administration of T. polium extract not only disclosed the significant anti-hyperglycemic potential of the plant, but also relieved dyslipidemia and oxidative stress related to diabetes. Furthermore, remarkable improvement of glucose tolerance was exhibited among the treated groups, confirming the presence of insulin in the blood. Subsequently, the effective molecular mechanism of T. polium was partially revealed by western blot analyses which detected considerable up-regulation of p-FoxO1 and Pdx1 proteins parallel to reduction of p-JNK expression among the treated diabetic rats. CONCLUSION: This study demonstrates that T. polium extract is able to restore the ß-cell mass and insulin secretion by regulation of pivotal transcription factor of the pancreatic ß-cells Pdx1 in JNK pathway.

Bcl6 gene-silencing facilitates PMA-induced megakaryocyte differentiation in K562 cells.

Targeted therapy via imatinib appears to be a promising approach for chronic myeloid leukemia (CML) therapy. However, refractory and resistance to imatinib therapy has encouraged many investigators to get involved in development of new therapeutic agents such as Phorbol 12-myrestrat 13-acetate (PMA) for patients with CML. In that line, we attempted to investigate the chemosensitizing effect of PMA on the imatinib-resistant cells. Based on our western blot analyses, resistant K562 cells (K562R) showed high levels of FoxO3a and Bcl6 expressions which were not modulated by imatinib treatment. However, upon PMA treatment, the levels of both FoxO3a and Bcl6 were up-regulated among both the sensitive and the resistant cells and this treatment was associated with initiation of megakaryocytic differentiation of the cells. SiRNA-silencing of FoxO3a led to augmentation of megakaryocytic differentiation of the cells. Similarly, siRNA gene silencing of Bcl6 enhanced the differentiation and induced cell apoptosis among both types of cells. Regarding these results, it might be concluded that Bcl6 knockdown combined with PMA therapy could present a new therapeutical strategy for refractory CML patients to imatinib.

Activation of STAT3/HIF-1α/Hes-1 axis promotes trastuzumab resistance in HER2-overexpressing breast cancer cells via down-regulation of PTEN.

BACKGROUND: Resistance to the HER2-targeted antibody trastuzumab remains to be a major clinical challenge in the treatment of HER2-positive breast cancer. Hyper-activation of STAT3 is proposed to be a predictive biomarker of trastuzumab resistance. However, the precise mechanism(s) remains poorly defined. Evidence is emerging that HIF-1α, a central downstream element of STAT3 pathway, serves a pivotal role in the complex signaling network with subsequent diverse cellular events. MATERIAL AND METHODS: We have established trastuzumab resistant SKBR3 cells (SKBR3-TR). The cell viability, apoptosis as well as western blot, siRNA transfection and co-immunoprecipitation assays were performed to evaluate the involvement of STAT3/HIF-1α in modulation of trastuzumab resistance. RESULTS: We found that in SKBR3-TR cells and conditioned medium-treated parental cells, constitutive phosphorylated STAT3 coincided with prominent up-regulation of HIF-1α which was accompanied with PTEN attenuation. Moreover, the inhibition of STAT3 activation by Stattic and/or genetically STAT3 knocking down decreased HIF-1α level in SKBR3-TR cells. Additionally, treatment with Stattic and/or STAT3 siRNA engendered the up-regulation of PTEN protein in STAT3-inhibited resistant cells. Restoration of PTEN was also observed following siRNA-mediated silencing of HIF-1α expression. Moreover, down-regulation of HIF-1α caused a reduction in the HES-1 content. Further study with HES-1 specific siRNA revealed the elevation of PTEN expression in HES-1 knock-down trastuzumab resistant cells. CONCLUSION: The impairment of STAT3-HIF-1α-HES-1 pathway restored trastuzumab sensitivity through up-regulation of PTEN protein. GENERAL SIGNIFICANCE: These findings highlighted the signal integrator role of HIF-1α in STAT3-mediated trastuzumab resistance induction which would be valuable in designing more efficient chemosensitization strategies.

Sensitizing effect of juglone is mediated by down regulation of Notch1 signaling pathway in trastuzumab-resistant SKBR3 cells.

Trastuzumab (Herceptin) monoclonal antibody directed against HER2 receptor has been administered as a treatment for metastatic HER2 positive breast cancer. The problematic issue in treatment of HER2 positive breast cancer cells is commonly the induction of resistance to trastuzumab which might be due to modulation of some vital signaling elements such as Notch1 and Pin1. In this study, we were aimed to investigate whether the cross talk between pin1 and Notch1 has a role in this event. Our results indicated that the expression level of Pin1 in resistant SKBR3 cells increased by about twofold relative to sensitive SKBR3 cells. Besides, Pin1 inhibition via juglone reduced the extent of proliferation, colony formation and migration capacity of resistant SKBR3 cells. In addition, despite a feed forward loop between Notch1 and Pin1 in sensitive SKBR3 cells, inhibition of Notch1 cleavage in resistant SKBR3 cells did not affect pin1 level whereas pin1 inhibition by juglone reduced the level of Hes1, p-Akt and increased the cellular content of Numb. Therefore, we concluded that pin1 inhibition could be considered as a promising sensitizing strategy to weaken trastuzumab resistance.

Mycophenolic acid potentiates HER2-overexpressing SKBR3 breast cancer cell line to induce apoptosis: involvement of AKT/FOXO1 and JAK2/STAT3 pathways.

Trastuzumab has been successfully used as a first-line therapy specific for HER2-overexressing breast cancer patients. However, despite the effectiveness of trastuzumab, the occurrence of inherent and acquired resistance remains as the main challenge of the therapy. Thus, this has motivated efforts toward finding new therapeutic strategies including combining trastuzumab with other drugs to enhance its therapeutic efficacy. In that line, we investigated the capability of mycophenolic acid (MPA), an inhibitor of de novo guanine nucleotide synthesis with potential anti-cancer activity, on improving the response to trastuzumab among SKBR3 cells as well as trastuzumab resistant SKBR3-TR cells. Our data indicated that irrespective to trastuzumab sensitivity of cells, MPA effectively inhibited cell growth through inducing adipocyte-like cell differentiation as well as blocking cell cycle progression at G1 phase along with augmentation of p27kip expression level. Furthermore, combined treatment with trastuzumab and MPA was more potent in cell growth inhibition, cell cycle arrest and apoptosis induction, as evident by flow cytometric analyses and caspase-3 production, in both trastuzumab sensitive and resistant SKBR3 cells. Besides, western blot analysis showed that elevated apoptosis induction in both cell groups was associated with attenuation in phosphorylation of some key elements of HER2 signaling pathway including AKT, ERK, STAT3 and consequently augmentation in FOXO1 expression level in response to combination of trastuzumab and MPA. These data suggest that manipulation of intracellular GTP level by MPA and consequent molecular perturbation in some of the cell survival and pro-apoptotic relevant signaling pathways might provide an alternative clinical strategy for chemosensitization of resistant breast cancer cells to anti- HER2 therapy.