Smad7 Protein Expression Research Articles

Tumor-derived TGF-β inhibits mitochondrial respiration to suppress IFN-γ production by human CD4+ T cells.

Transforming growth factor-β (TGF-β) is produced by tumors, and increased amounts of this cytokine in the tumor microenvironment and serum are associated with poor patient survival. TGF-β-mediated suppression of antitumor T cell responses contributes to tumor growth and survival. However, TGF-β also has tumor-suppressive activity; thus, dissecting cell type-specific molecular effects may inform therapeutic strategies targeting this cytokine. Here, using human peripheral and tumor-associated lymphocytes, we investigated how tumor-derived TGF-β suppresses a key antitumor function of CD4+ T cells, interferon-γ (IFN-γ) production. Suppression required the expression and phosphorylation of Smad proteins in the TGF-β signaling pathway, but not their nuclear translocation, and depended on oxygen availability, suggesting a metabolic basis for these effects. Smad proteins were detected in the mitochondria of CD4+ T cells, where they were phosphorylated upon treatment with TGF-β. Phosphorylated Smad proteins were also detected in the mitochondria of isolated tumor-associated lymphocytes. TGF-β substantially impaired the ATP-coupled respiration of CD4+ T cells and specifically inhibited mitochondrial complex V (ATP synthase) activity. Last, inhibition of ATP synthase alone was sufficient to impair IFN-γ production by CD4+ T cells. These results, which have implications for human antitumor immunity, suggest that TGF-β targets T cell metabolism directly, thus diminishing T cell function through metabolic paralysis.

Protective Effects of Sesamin against UVB-Induced Skin Inflammation and Photodamage In Vitro and In Vivo.

Ultraviolet (UV) exposure has been demonstrated as the most critical factor causing extrinsic skin aging and inflammation. This study explored the protective effects and mechanisms of sesamin against skin photodamage. Sesamin reduced intracellular reactive oxygen species production after UVB irradiation in human dermal fibroblasts. The sesamin treatment attenuated mitogen-activated protein (MAP) kinase phosphorylation and matrix metalloproteinase (MMPs) overexpression induced by UVB exposure, and it significantly enhanced the tissue inhibitor of metalloproteinase-1 protein expression. Sesamin also elevated the total collagen content in human fibroblasts by inhibiting UVB-induced mothers against decapentaplegic homolog 7 (Smad7) protein expression. Sesamin reduced UVB-induced inducible nitric oxide synthase (i-NOS) and cyclooxygenase-2 (COX-2) overexpression and inhibited nuclear factor-kappa B (NF-κB) translocation. Moreover, sesamin may regulate the c-Jun N-terminal kinases (JNK) and p38 MAP kinase pathways, which inhibit COX-2 expression. Sesamin could reduce UVB-induced inflammation, epidermal hyperplasia, collagen degradation, and wrinkle formation in hairless mice. It also reduced MMP-1, interleukin (IL-1), i-NOS, and NF-κB in the mouse skin. These results demonstrate that sesamin had antiphotodamage and anti-inflammatory activities. Sesamin has potential for use as a skin protection agent in antiphotodamage and skin care products.

Hsa-miR-346 plays a role in the development of sepsis by downregulating SMAD3 expression and is negatively regulated by lncRNA MALAT1

Sepsis is a common complication in infection, trauma, and surgery. Severe sepsis has been identified as the leading cause of death in patients suffering from noncardiovascular ailments in intensive care units. In the current study, we used lipopolysaccharide (LPS) to stimulate the mouse macrophage cell line RAW264.7, and investigated the effects of the lncRNA MALAT1/hsa-miR-346/SMAD3 regulatory network on the progression of sepsis. We showed that MALAT1 inhibited RAW264.7 cell proliferation, while hsa-miR-346 promoted its proliferation. In this RAW264.7 cell model, MALAT1 inhibited hsa-miR-346 expression, and upregulated SMAD3 protein expression. The SMAD3 protein expression in RAW264.7 cells was significantly downregulated upon the overexpression of hsa-miR-346. These results suggest that the MALAT1/hsa-miR-346/SMAD3 regulatory network plays a key role in the development of sepsis, and may serve as a target for the treatment of sepsis.

TGF-β signaling proteins and CYP24A1 may serve as surrogate markers for progesterone calcitriol treatment in ovarian and endometrial cancers of different histological types

Background: Strategies are needed to coordinately block drivers and induce suppressors of cancer to reduce incidence and improve outcomes for individuals with inherited or acquired risk. We previously reported the chemopreventive and therapeutic efficacy of the combination of progestin and calcitriol in transformed and malignant endometrioid endometrial cancer (EC) and in ovarian cancer models involving attenuated expression of TGF-β signaling proteins and progestin-mediated inhibition of calcitriol-induced CYP24A1 expression. This study aims to expand the applications for this combination to other subtypes of endometrial and ovarian cancers, including those with mutations in ARID1A or PIK3CA, DNA mismatch repair (MMR) deficiency or BRCA1 null status. Methods: Ovarian and EC cell lines of different histotypes were cultured with either progesterone, calcitriol, or the combination of progesterone and calcitriol for 3 or 5 days. The end points for this in vitro investigation included assessments of cell growth by (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assays and the expression of TGF-β ligands, receptors, SMAD proteins and CYP24A1 by western blotting. Results: Treatment of ovarian clear cell carcinoma, endometrioid carcinoma, papillary serous adenocarcinoma, BRCA1 null, and DNA MMR deficient EC cell lines with progesterone alone or in combination with calcitriol inhibited cell growth and expression of TGF-β1, TGF-β2, TGF-Rβ1, TGF-βR2, pSMAD2/3 and CYP24A1. Expression of TGF-βR3, SMAD-4, progesterone receptor (PR) and vitamin-D receptor (VDR) was not altered in any cell line tested except, ES-2, where VDR expression was upregulated in response to treatment. Conclusions: These results suggest that progesterone alone and progesterone-calcitriol combination have broad application in both chemopreventive and therapeutic settings that merit further development in a wide variety of ovarian and ECs, including those derived from germline or somatic mechanisms. Moreover, our data suggest that TGF-β signaling proteins and CYP24A1 may be effective surrogate markers indicative of treatment response.

Astragaloside IV inhibits excessive mesangial cell proliferation and renal fibrosis caused by diabetic nephropathy via modulation of the TGF‑β1/Smad/miR‑192 signaling pathway

Astragaloside IV (ASI) exhibits a wide variety of pharmacological effects in cardiovascular diseases, hepatitis and kidney disease and due to this, ASI has recently become an attractive research target. The present study aimed to determine the effect of ASI on renal fibrosis and the mechanisms underlying its therapeutic effects in diabetic nephropathy (DN). In vitro, ASI was added to rat mesangial cells (RMCs) and cultured with a high level of glucose (HG) to observe the effects exhibited on proliferation and fibrosis-related mRNA and protein expression. In vivo, a DN model was established using streptozotocin administration in rats, and renal injury was evaluated using renal histological examination. The expression levels of related mRNAs and proteins were analyzed using reverse transcription-quantitative PCR, western blot analysis and immunohistochemistry. ASI was demonstrated to downregulate miR-192 expression and inhibit excessive proliferation of RMCs, which was induced by HG, in a dose-dependent manner. Additionally, ASI exhibited a therapeutic effect on DN rats. ASI was also demonstrated to decrease the miR-192 expression and mRNA and protein expression of transforming growth factor-β1 (TGF-β1), Smad3, α-smooth muscle actin (α-SMA) and collagen type 1 (col1), and increase the mRNA and protein expression of Smad7 in vitro and in vivo. These results suggested that ASI exhibited a therapeutic effect on DN, possibly due to the inhibition of excessive mesangial proliferation and renal fibrosis via the TGF-β1/Smad/miR-192 signaling pathway.

Loss of SMAD4 protein expression in gastrointestinal and extra-gastrointestinal carcinomas.

SMAD4 (DPC4) is a tumour suppressor gene that is dysregulated in various tumour types, particularly pancreaticobiliary and gastrointestinal carcinomas. Corresponding loss of protein expression has been reported in approximately 50% of pancreatic and 25% of colonic adenocarcinomas. In the evaluation of carcinoma of unknown primary site, immunohistochemical loss of SMAD4 expression is often used to suggest pancreaticobiliary origin, but there are limited data on the spectrum of SMAD4 expression in carcinomas of other sites. This study evaluates the frequency of SMAD4 loss in a large cohort of carcinomas from diverse anatomical sites. Immunohistochemistry for SMAD4 was performed on tissue microarrays or whole tissue sections of 1210 carcinomas from various organs: gastrointestinal tract, liver, pancreas/biliary tract, lung, breast, thyroid, kidney, ovary and uterus. Expression was considered lost when there was complete absence of staining in tumour cell nuclei, in the presence of intact staining in non-neoplastic cells. SMAD4 loss was seen in 58% of pancreatic adenocarcinomas, 27% of appendiceal adenocarcinomas, 19% of colorectal adenocarcinomas, 16% of cholangiocarcinomas, 10% of lung adenocarcinomas and <5% of oesophageal, breast, gastric and mucinous ovarian adenocarcinomas. All papillary thyroid, hepatocellular, non-mucinous ovarian, endometrial and renal cell carcinomas showed intact SMAD4 nuclear expression. In addition to pancreaticobiliary, appendiceal and colonic tumours, SMAD4 loss is also seen in a small subset of other carcinomas, specifically breast, lung, oesophageal and gastric adenocarcinomas, all of which are typically CK7-positive, similar to pancreaticobiliary carcinoma. Awareness of SMAD4 loss in these other carcinoma types is helpful in the evaluation of carcinomas of unknownor uncertain primary site.

Mechanism of miR-15a regulating the growth and apoptosis of human knee joint chondrocytes by targeting SMAD2

Objective To investigate the effects of miR-15a on proliferation and apoptosis of human knee articular chondrocytes and explore its underlying mechanism. Methods qRT-PCR was used to detect the expression of miR-15a in normal chondrocytes and knee arthritic chondrocytes; miR-con (transfected miR-con), miR-15a (transfected miR-15a mimics), anti-miR-con group (transfected anti-miR-con), anti-miR-15a group (transfected anti-miR-15a mimics), pcDNA group (transfected pcDNA), pcDNA-SMAD2 group (transfected pcDNA-SMAD2), the miR-15a + pcDNA group (co-transfected miR-15a and pcDNA), miR-15a + pcDNA-SMAD2 group (co-transfected miR-15a mimics and pcDNA-SMAD2), were transfected into knee articular chondrocytes by liposome method, respectively. The cell proliferation and apoptosis of each group were detected by MTT assay and flow cytometry. The protein expression of SMAD2 was detected by Western blot. The fluorescence activity of each group was detected by dual luciferase reporter gene assay. Results The expression of miR-15a in knee arthritis chondrocytes was significantly increased (p < .05) compared with that in normal chondrocytes. Moreover, overexpression of miR-15a and silencing of SMAD2 inhibited proliferation and promoted apoptosis in knee arthritis chondrocyte. MiR-15a targeted SMAD2. Overexpression of SMAD2 reversed the inhibitory effects on proliferation and promotion effects on apoptosis induced by miR-15a in knee arthritis chondrocytes. Conclusion miR-15a can inhibit the proliferation and promote apoptosis of knee arthritis chondrocytes. The mechanism may be related to SMAD2, which will provide a new target for the treatment of knee arthritis.

Overexpression of SOX9 alleviates the progression of human osteoarthritis in vitro and in vivo.

PurposeRecent findings have identified that SOX9 served as a key role during the pathogenesis of osteoarthritis (OA). This study aimed to investigate the mechanisms by which SOX9 regulated the formation of OA in vitro and in vivo.Materials and methodsThe relative expressions of SOX9 in patients with OA and normal fracture of thighbone were analyzed by real-time-PCR. In vitro, IL-1β induced inflammatory response in human chondrocytes was used to evaluate the function of SOX9. The recombinant SOX9 lentivirus vector (Lenti-SOX9) was used to upregulate the expression of SOX9 in cells. ELISA was used to measure the concentration of tumor necrosis factor-α (TNF-α). The protein expressions of SOX9, matrix metalloproteinase-13 (MMP13), Collagen II, Aggrecan and Smad3 were analyzed by Western blot. Cell proliferation and cell apoptosis were detected by CCK-8 assay and flow cytometry, respectively. In vivo, the effect of SOX9 on surgically induced OA mice was evaluated.ResultsThe gene level of SOX9 was remarkably downregulated in patients with OA compared with normal people, while the concentration of TNF-α was upregulated. In addition, IL-1β reduced the expressions of SOX9, Collagen II and Aggrecan and increased the level of MMP13 in chondrocytes. Moreover, Lenti-SOX9 notably inhibited IL-1β-induced growth inhibition and apoptosis in chondrocytes via increasing the expression of Smad3. Finally, Lenti-SOX9 markedly alleviated the symptoms of OA mice in vivo.ConclusionUpregulation of SOX9 inhibited IL-1β-induced inflammatory response via increasing the level Smad3 in human chondrocytes and exhibited therapeutic effect on surgically induced OA mice in vivo. Therefore, SOX9 may serve as a potential target in the treatment of OA in the future.

TGF-β signaling proteins and CYP24A1 may serve as surrogate markers for progesterone calcitriol treatment in ovarian and endometrial cancers of different histological types.

BackgroundStrategies are needed to coordinately block drivers and induce suppressors of cancer to reduce incidence and improve outcomes for individuals with inherited or acquired risk. We previously reported the chemopreventive and therapeutic efficacy of the combination of progestin and calcitriol in transformed and malignant endometrioid endometrial cancer (EC) and in ovarian cancer models involving attenuated expression of TGF-β signaling proteins and progestin-mediated inhibition of calcitriol-induced CYP24A1 expression. This study aims to expand the applications for this combination to other subtypes of endometrial and ovarian cancers, including those with mutations in ARID1A or PIK3CA, DNA mismatch repair (MMR) deficiency or BRCA1 null status.MethodsOvarian and EC cell lines of different histotypes were cultured with either progesterone, calcitriol, or the combination of progesterone and calcitriol for 3 or 5 days. The end points for this in vitro investigation included assessments of cell growth by (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assays and the expression of TGF-β ligands, receptors, SMAD proteins and CYP24A1 by western blotting.ResultsTreatment of ovarian clear cell carcinoma, endometrioid carcinoma, papillary serous adenocarcinoma, BRCA1 null, and DNA MMR deficient EC cell lines with progesterone alone or in combination with calcitriol inhibited cell growth and expression of TGF-β1, TGF-β2, TGF-Rβ1, TGF-βR2, pSMAD2/3 and CYP24A1. Expression of TGF-βR3, SMAD-4, progesterone receptor (PR) and vitamin-D receptor (VDR) was not altered in any cell line tested except, ES-2, where VDR expression was upregulated in response to treatment.ConclusionsThese results suggest that progesterone alone and progesterone-calcitriol combination have broad application in both chemopreventive and therapeutic settings that merit further development in a wide variety of ovarian and ECs, including those derived from germline or somatic mechanisms. Moreover, our data suggest that TGF-β signaling proteins and CYP24A1 may be effective surrogate markers indicative of treatment response.

Genomic alterations in signet ring and mucinous patterned colorectal carcinoma

BackgroundThe genetic alterations (GAs) in two specific histological subtypes of colorectal cancer (CRC), signet ring cell colorectal carcinoma (SRC) and mucinous colorectal carcinoma (MC), are not well known. In the present study, we employed next-generation sequencing to perform genetic profiling of SRC and MC, and compared the spectrum of GAs with the alterations found in conventional type colorectal cancer (CON). Materials and methodsWe selected 46 CRCs comprising 17 SRCs and mucinous carcinoma with signet ring cell component (SRCCs), 17 MCs, and 12 CONs with microsatellite stability or microsatellite instability-low. Deep sequencing was performed using a targeted cancer panel composed of 171 cancer-related genes. SMAD4 protein expression was evaluated by immunohistochemical staining. ResultsWe detected 108 mutations in 18 different genes. Overall, 2.34 GAs were detected per tumor (range, 0–14). The overall frequency of GA and alteration in targetable genes was less prevalent in SRC/SRCC compared to the frequency of alteration in MC/CON (p = 0.040 and p < 0.001, respectively). The GA profile of SRC/SRCC included TP53 (8/17, 47.1%), SMAD4 (5/17, 29.4%), KRAS (4/17.23.5%), APC (4/17.23.5%), PIK3CA, ATM, BRAF, and PIK3R1 (1/17, 5.9%, each). KRAS mutation was significantly less prevalent in SRC/SRCC compared to the number of KRAS mutations in MC (12/17, 70.6%) and CON (9/12, 75.0%) (p = 0.015 and 0.01, respectively). Compared to the 152 non-hypermutated CONs from TCGA database, SMAD4 alteration was predominant in SRC/SRCC (p = 0.045) with aberrant loss of SMAD4 expression (13/17, 76.5%) compared to the SMAD4 alterations in CON (5/15, 33.3%) (p = 0.031). Accordingly, KRAS (12/17, 70.6%), APC (6/17, 35.3%), SMAD4, TP53 (4/17, 23.5%, each), PIK3CA (3/17, 17.6%), AKT1, ATM, BRAF, EGFR, and EZH2 (1/17, 5.9%, each) were altered in MC. APC and TP53 mutations were less frequent in MC compared to those in TCGA-CON (p < 0.001 and 0.003, respectively) whereas KRAS mutation was prevalent (p = 0.041). ConclusionAlterations of known cancer associated genes and targetable genes in SRC/SRCC are infrequent. The profile of GAs in SRC/SRCC and MC differs from the GA profile of CON. Specifically, SMAD4 mutation and loss of SMAD4 expression is frequently found in SRC/SRCC. The genetic profiles revealed in the present study may aid in developing precision medicine for CRC treatment based on histological subtype.

Effects of bone morphogenetic protein 2 on ossification of posterior longitudinal ligament and possible mechanism

Objective To investigate the effect of bone morphogenetic protein 2(BMP2)on ossification of the posterior longitudinal ligament (OPLL) and its relationship with transforming growth factor-β (TGF-β)/Smad signaling pathway. Methods The expression vectors of wild type pcDNA3.1-BMP2 (WT), mutant pcDNA3.1-BMP2 (37G), mutant pcDNA3.1-BMP2 (190T) and mutant pcDNA3.1-BMP2 (37G/190T) were constructed and identified by agarose gel electrophoresis. The constructed vector was transfected into mouse embryonic fibroblasts C3H10T1/2 mediated by liposome to detect the expression of BMP2. Six groups were divided according to the transfection situation: (1) the non-transfection group; (2) empty vector pcDNA3.1 transfection group; (3) pcDNA3.1-bmp2 (WT) transfection group; (4) pcDNA3.1-bmp2 (37G) transfection group; (5) pcDNA3.1-bmp2 (190T) transfection group; (6) pcDNA3.1-bmp2 (37G/190T) transfection group. The experimental and control group were defined according to whether BMP2 polymorphism was included. Therefore, the non-transfection group and empty vector pcDNA3.1 transfection group were control groups, and the other groups were experimental groups. The expression of phosphorylated Smad1/5/8 and Smad4 in positive cell clones were detected by western blotting, and the alkaline phosphatase (ALP) was detected by quantitative detection kits. The protein expressions were compared among the experimental groups. Results Two fragments digested from pcDNA3.1-BMP2 represented 1.2 kb and 5.4 kb by agarose electrophoresis. The direct sequencing results were in accordance with target gene sequence. BMP2 gene was successfully transfected and stably expressed in C3H10T1/2 cells. Western blotting showed that the expression of phosphorylated Smad1/5/8 protein in the experimental groups was increased significantly after transfection, with significant difference between the experimental groups and the control groups (P 0.05). The expressions of Smad4 protein transfected by wild or mutation type pcDNA3.1-BMP2 were significantly higher than those in the control groups (P 0.05). However, there were significant differences between the two groups and other experimental groups (P<0.05). The Ser37Ala (T/G) polymorphism in exon 2 of BMP2 gene was positively correlated with ALP activity in stably transfected C3H10T1/2 cells. Conclusion The Ser37Ala (T/G) polymorphism in exon 2 of BMP2 gene promotes OPLL ossification through TGF-β/Smad signaling pathway, the possible mechanism for which is to up-regulate the protein expressions of Smad4 and ALP. Key words: Ossification, posterior longitudinal ligament; Bone morphogenetic protein 2; Signal transduction; Single nucleotide polymorphisms

Abstract 2976: A comparative study between the therapeutic efficacy of vitamin D3 and its analogue, paricalcitol, in the treatment of colon cancer in rat

Abstract Background: Vitamin D3 (VD) and its analogue, Paricalcitol (Pcal), have previously shown anti-tumorigenic activities against colorectal cancer (CRC). Hence, this study compared the therapeutic efficacy of both VD types with or without chemotherapy (5-FU) in azoxymethane (AOM)-induced CRC in rat. Methods: seventy male Wistar rats were equally allocated into 7 groups: Control, AOM, VD, Pcal, 5-FU, 5-FU/VD and 5-FU/Pcal. AOM was subcutaneously injected for 2 weeks (15mg/kg/week). The study duration was 25 weeks. VD (500 IU/rat/day; 3 days/week) and Pcal (2.5 μg/kg/day; 3 days/week) were injected subcutaneously from week-15 till the end of the study. 5-FU intraperitoneal injections started in week-21 (12 mg/kg/day; 4 successive days) and continued in week-22 (6 mg/kg/4 doses every other day). The colons were collected for gross and histopathological examination. The expression of VD signalling system, Wnt, β-catenin, NF-κB, COX-2, iNOS, VEGF, smad4, p21 and p27 proteins was measured by immune-histochemistry/fluorescence. The mRNA expression of Wnt, β-catenin, DKK-1 and COX-2 genes was measured by Quantitative RT-PCR. ELISA was used to measure the concentrations of TGF-β1, VEGF and COX-2 proteins in tissues. Results: The AOM and 5-FU groups showed marked inhibitions in the VD receptor and binding protein compared with controls (P &amp;lt; 0.05). VD and Pcal monotherapies significantly increased the expression of the VD molecules and the VD group showed the utmost elevations. Moreover, the tumour numbers and sizes were equally and significantly reduced in the 5-FU, VD and Pcal groups compared with the AOM group (P &amp;lt; 0.05). Interestingly, the 5-FU/VD and 5-FU/Pcal resulted in significantly lower numbers of tumours compared with the remaining study groups and the highest significant reduction was observed in the former group. At the molecular level, the 5-FU/VD markedly inhibited the pro-oncogenic molecules (Wnt, β-catenin, NF-κB, COX-2, iNOS &amp; VEGF) as well as significantly upregulated the anti-tumorigenic molecules (DKK-1, TGF-β1, smad4, p21 and p27) compared with the other study groups (P &amp;lt; 0.05). Conclusions: Vitamin D3 showed a better synergism than Pcal with 5-FU. The enhanced anti-tumorigenic actions of 5-FU/VD involved the modulation of the Wnt/β-catenin and TGF-β1/smad4 pathways, angiogenesis and cell cycle arrest. Further studies are required to elucidate the therapeutic significance of VD in clinical settings Citation Format: Mohammed Abbas Baghdadi, Mohammed Aslam, Jawwad Ahmad, Shakir Idris, Bassem Refaat. A comparative study between the therapeutic efficacy of vitamin D3 and its analogue, paricalcitol, in the treatment of colon cancer in rat [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2976.

Abstract 4921: TGF-beta signaling proteins and CYP24A1 may serve as surrogate markers for progesterone calcitriol treatment in ovarian and endometrial cancers of different histological types

Abstract Strategies are needed to coordinately block drivers and induce suppressors of cancer to reduce incidence and improve outcomes for individuals with inherited or acquired risk. We previously reported the chemopreventive and therapeutic efficacy of the combination of progestin and calcitriol in transformed and malignant endometrioid endometrial cancer and in ovarian cancer models involving attenuated expression of TGF-β signaling proteins and progestin-mediated inhibition of calcitriol-induced CYP24A1 expression. This study aims to expand the applications for this combination to other subtypes of endometrial and ovarian cancers including those with mutations in ARID1A or PIK3CA, DNA mismatch repair deficiency or BRCA1-null status. The end points for this in vitro investigation included assessments of cell growth and the expression of TGF-β ligands, receptors, SMAD proteins and CYP24A1. Treatment of ovarian clear cell carcinoma, endometrioid carcinoma, papillary serous adenocarcinoma, BRCA1 null, and DNA mismatch repair deficient endometrial cancer cell lines with progesterone alone or in combination with calcitriol inhibited cell growth and expression of TGF-β1, TGF-β2, TGF-Rβ1, TGF-βR2, pSMAD2/3 and CYP24A1. Expression of TGF-βR3, SMAD-4, PR and VDR was not altered in any cell line tested except, ES-2, where VDR expression was upregulated in response to treatment. These results suggest that progesterone alone and progesterone-calcitriol combination have broad application in both chemopreventive and therapeutic settings that merit further development in a wide variety of ovarian and endometrial cancers, including those derived from germline or somatic mechanisms. Moreover, our data suggest that TGF-β signaling proteins and CYP24A1 may be effective surrogate markers indicative of treatment response. Citation Format: Ana Paucarmayta, Hannah Taitz, Yovanni Casablanca, Gustavo C. Rodriguez, G L. Maxwell, Kathleen M. Darcy, Viqar Syed. TGF-beta signaling proteins and CYP24A1 may serve as surrogate markers for progesterone calcitriol treatment in ovarian and endometrial cancers of different histological types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4921.

Abstract 4286: TGFβ induced SMAD4 dependent apoptosis proceeded by EMT in colorectal cancer

Abstract Colorectal cancer (CRC) is one of the leading cause of cancer-related deaths Worldwide. In Saudi Arabia, CRC is more aggressive and presents at younger age, warranting new treatment strategies. Role of TGFβ/Smad4 signaling pathway in initiation and progression of CRC is well documented. Current study examined the role of TGFβ/Smad4 signaling pathway in a large cohort of Saudi CRC, followed by in vitro analysis to dissect the dual role of TGFβ on inducing epithelial to mesenchymal transition (EMT) and apoptosis. In this study, we investigated Smad4 alterations and their association with clinicopathological outcomes in a large cohort of CRC samples using targeted capture sequencing, Fluorescent in-situ hybridisation and immunohistochemistry. Our study demonstrated high frequency of Smad4 alterations with low expression of Smad4 protein identifying a sub-group of aggressive CRC to be an independent marker for poor prognosis. Functional studies using CRC cells show that TGFβ induces Smad4 dependent EMT followed by apoptosis. Induction of mesenchymal transcriptional factors, Snail1 and Zeb1 was essential for TGFβ-induced apoptosis. Our results indicate that KLF5 acts as an oncogene in CRC cells regardless of Smad4 expression and inhibition of KLF5 is requisite for TGFβ-induced apoptosis. Furthermore, TGFβ/Smad4 signal inhibits the transcription of KLF5 that in turn switches Sox4 from tumor promoter to suppressor. A high incidence of Smad4 alterations were found in the Saudi CRC patients. Functional study results indicate that TGFβ induces Smad4 dependent EMT followed by apoptosis in CRC cells. Citation Format: Pratheeshkumar Poyil, Abdul K. Siraj, Divya Sasidharan Padmaja, Sandeep Kumar Parvathareddy, Rong Bu, Tariq Masoodi, Yan Kong, Saravanan Thangavel, Saeeda Omer Ahmed, Nasser Al-Sanea, Luai H. Ashari, Alaa Abduljabbar, Samar Alhomoud, Fouad Al-Dayel, Khawla S. Al-Kuraya. TGFβ induced SMAD4 dependent apoptosis proceeded by EMT in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4286.

TGFβ-induced SMAD4-dependent Apoptosis Proceeded by EMT in CRC.

Colorectal cancer is one of the leading causes of cancer-related deaths worldwide. In Saudi Arabia, colorectal cancer is more aggressive and presents at younger age, warranting new treatment strategies. Role of TGFβ/Smad4 signaling pathway in initiation and progression of colorectal cancer is well documented. This study examined the role of TGFβ/Smad4 signaling pathway in a large cohort of Saudi patients with colorectal cancer, followed by in vitro analysis to dissect the dual role of TGFβ on inducing epithelial-to-mesenchymal transition (EMT) and apoptosis. Our study demonstrated high frequency of Smad4 alterations with low expression of Smad4 protein identifying a subgroup of aggressive colorectal cancer to be an independent marker for poor prognosis. Functional studies using colorectal cancer cells show that TGFβ induces Smad4-dependent EMT followed by apoptosis. Induction of mesenchymal transcriptional factors, Snail1 and Zeb1, was essential for TGFβ-induced apoptosis. Our results indicate that KLF5 acts as an oncogene in colorectal cancer cells regardless of Smad4 expression and inhibition of KLF5 is requisite for TGFβ-induced apoptosis. Furthermore, TGFβ/Smad4 signal inhibits the transcription of KLF5 that in turn switches Sox4 from tumor promoter to suppressor. A high incidence of Smad4 alterations were found in the Saudi patients with colorectal cancer. Functional study results indicate that TGFβ induces Smad4-dependent EMT followed by apoptosis in colorectal cancer cells.

Bone marrow mesenchymal stem cells regulate TGF-β to adjust neuroinflammation in postoperative central inflammatory mice.

Postoperative cognitive dysfunction (POCD) is one of the common postoperative complications, which is more common in aged patients. POCD mainly manifests as cognitive function changes after surgery, such as memory decline and inattention. In some severe cases, patients may suffer from personality changes and (or) social behavior decline. The aim of the current study is to confirm the effect and elucidate the mechanism of bone marrow mesenchymal stem cells (BMSCs) in postoperative central inflammatory mice. Mice were randomly assigned to four groups: sham, sham+BMSCs, model, and BMSCs group. In the model group, mice were intraperitoneally injected 8 mg/kg per day lipopolysaccharide for 5 days. In sham+BMSCs and BMSCs group, BMSCs (1 × 10 7 ) in 100 µL saline were injected into sham mice and model mice, respectively. In the model group, transforming growth factor β (TGF-β) protein expression was significantly increased, compared with that in the sham group. BMSCs were treated into postoperative central inflammatory mice, which resulted in a decreased of TGF-β protein expression. TGF-β and smad2 protein expression were suppressed, and apoptosis rate and inflammation were inhibited in coculture with BMSCs. The suppression of TGF-β inhibited the effects of BMSCs on apoptosis rate and inflammation in postoperative central inflammatory through a smad2 signaling pathway. The promotion of TGF-β reduced the effects of BMSCs on apoptosis rate and inflammation in postoperative central inflammatory through a smad2 signaling pathway. The present study demonstrates that BMSCs regulates TGF-β to adjust neuroinflammation in postoperative central inflammatory mice.

Effcets of mesenchymal stromal cell-derived exosomes deliver microRNA-145-5p on pancreatic ductal adenocarcinoma progression

Objective To investigate whether human umbilical cord mesenchymal stem cells-derived exosomes could inhibit pancreatic ductal adenocarcinoma progression. Methods The human umbilical cord derived-mesenchymal stem cells (huc-MSCs) loaded with microRNA (miRNA, miR)-145-5p mimics and negative control miRNA were added to pancreatic ductal adenocarcinoma (PDAC) cells. The proliferative activity and invasion abilities of BxPC3 and PANC-1 cells were measured by cell counting kit-8 (CCK-8) and Transwell assays. The cell cycle and cell apoptosis were detected by flow cytometry analysis. The expression levels of Smad3 were detected by Western blotting. Results HucMSC exosomes were isolated and purified from the hucMSC-conditioned medium by ultracentrifugation. HucMSC exosomes had a characteristic 'saucer-like’ morphology with diameters that ranged from 52.5 nm to 185.5 nm, with an average of 119 nm and the concentration of hucMSC exosomes was 400 Particles/ml. Further results showed positive expression of exosomal markers, such as CD9 and CD81. The results of cell proliferation showed that the absorbance value of BxPC3 and PANC-1 cells in 145-exo group was significantly lower than that of the NC-exo group after 4 days [(2.681±0.049) vs. (3.683±0.003), t=20.460, P<0.05; (2.646±0.116) vs. (3.450±0.112), t=4.992, P<0.05]. The results of Transwell assays showed that the number of metastatic ceils in the 145-exo group was significantly decreased compared to the NC-exo group [(68.200±3.262) cells vs. (104.800±2.177) cells, t=9.333, P<0.05; (83.000±3.493) cells vs. (146.200±4.432) cells, t=11.200, P<0.05]. Flow cytometric analysis revealed that 145-exo group caused an increase in the percentage of the cells in the G0/G1 stage of the cell cycle compared to the NC-exo group [(43.030±1.060)% vs. (31.730±0.843)%, t=8.346, P<0.05)]. Flow cytometric analysis revealed that 145-exo group increase in apoptotic cells, relative to NC-exo group [(10.970±0.405)% vs. (6.863±0.099)%, t=9.830, P<0.05; (2.400±0.166)% vs. (1.513±0.045)%, t=5.167, P<0.05]. The expression of Smad3 protein in 145-exo group was reduced by 38.0% and 37.4%, respectively (t=6.362, P<0.05; t=11.430, P<0.05). Conclusion HucMSCs-exo can effectively deliver miR-145-5p to inhibit pancreatic ductal adenocarcinoma progression, which may be related to downregulation of Smad3 expression. Key words: Human umbilical cord mesenchymal stromal cell; Exosomes; MicroRNA-145-5p; PANCreatic ductal adenocarcinoma; Treatment

Effect of paeoniflorin on cardiac remodeling in chronic heart failure rats through the transforming growth factor β1/Smad signaling pathway.

Cardiac remodeling is an important mechanism for the occurrence and development of chronic heart failure (CHF). Paeoniflorin (Pae) is the main active ingredient of Chinese herbaceous peony and has novel anti-inflammatory effect. This study was conducted to assess the effects and mechanisms of Pae on cardiac remodeling in CHF rats. A cardiac remodeling rat model was induced by isoprenaline (Iso). Pae (20 µg/kg/d) was administrated to CHF rats for six weeks. Cardiac ultrasound was used to assess the structure and function of CHF rats. Collagen volume fraction (CVF) and perivascular collagen volume area of myocardial tissues were calculated. With real-time polymerase chain reaction and Western blot, the protein and mRNA levels of transforming growth factor β1 (TGF-β1) and Smad3 were detected. Compared to Iso group, Pae can alleviate cardiac remodeling and improve cardiac function in CHF rats. The levels of CVF and perivascular collagen volume area reduced in Pae group (P<0.05). The expression of TGF-β1 and Smad3 protein decreased in Pae and Cap group (P<0.05). Further, the expression of TGF-β1 and Smad3 mRNA also decreased markedly in the Pae group (P<0.05). Pae could attenuate cardiac remodeling and improve cardiac function in CHF rats. The potential mechanism for the cardioprotective effect of Pae may be highly associated with the down-regulating of TGF-β1/Smad signaling pathway.

Corrigendum] Human peripheral blood‑derived exosomes for microRNA delivery.

Exosomes serve important functions in cell‑to‑cell communication and biological functions by serving as a delivery cargo shuttle for various molecules. The application of an improved delivery method for microRNAs (miRNAs/miRs) may enhance their potential as a therapeutic tool in cardiac diseases. Thus, the present study investigated whether human peripheral blood‑derived exosomes may be used as a delivery cargo system for miRNAs, and whether the delivery of miR‑21 using a human peripheral blood derived‑exosome may influence the degree of remodeling following myocardial infarction (MI). In H9C2 and HL‑1 cells, miR‑21 expression was successfully regulated by treatment with human peripheral blood derived‑exosomes loaded with an miR‑21 mimic or inhibitor compared with untreated cells. In addition, the mRNA and protein expression levels of SMAD family member7 (Smad7), phosphatase and tensin homolog (PTEN) and matrix metalloproteinase 2 (MMP2), which are involved in cardiac fibrosis, were associated with the uptake of miR‑21 mimic‑ or inhibitor‑loaded exosomes. Similarly, the invivo mRNA and protein expression of Smad7, PTEN and MMP2 were altered following treatment with miR‑21 mimic‑ or inhibitor‑loaded exosomes. Furthermore, miR‑21 mimic‑loaded exosomes enhanced fibrosis, whereas miR‑21 inhibitor‑loaded exosomes reduced fibrosis in a mouse MI model. These results suggested that miRNA‑loaded human peripheral blood derived‑exosomes may be used as a therapeutic tool for cardiac diseases. [the original article was published in International Journal of Molecular Medicine 43: 2319‑2328, 2019; DOI:10.3892/ijmm.2019.4150].

PARP1 Inhibition as a Novel Therapeutic Target for Keloid Disease.

Objective: Inactivation of poly(ADP-ribose) polymerase 1 (PARP1) has been found to have protective effect in several fibrotic diseases. But the effect is not studied yet in keloids. Herein, we evaluated the therapeutic effect of PARP1 inhibitor, rucaparib, for keloids.Approach: The protein expressions of PARP1 and smad3 were evaluated with western blotting in keloids and controls. The effect of rucaparib was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and migration assay. We further analyzed the effect of rucaparib on patient-derived keloid xenograft murine model.Results: The protein expressions of PARP1 and smad3 were significantly higher in keloid tissue. Rucaparib (20 μM) significantly suppressed the proliferation of keloid fibroblasts. Moreover, the combination of rucaparib (20 μM) and triamcinolone (50 μM) showed additive suppressive effect on keloid fibroblasts. Migration assay showed that rucaparib (10 μM) significantly suppressed the migration of keloid fibroblasts. Fibrosis markers in keloid fibroblasts significantly decreased after rucaparib treatment (20 μM). In patient-derived keloid xenograft model, rucaparib significantly reduced the size of keloid tissue.Innovation and Conclusion: The study data suggest PARP1 might be a novel therapeutic target for keloid disease. PARP1 inhibitor, rucaparib, might be a promising therapeutic drug for the treatment of keloid disease.