Cytotoxicity of curcumin against CD44± prostate cancer cells: Roles of miR-383 and miR-708.

Objective: Cancer stem cells (CSCs) remaining in the tumor tissues after applying treatments may cause recurrence or metastasis of prostate cancer (PC). Curcumin has the promising potential to target CSCs. Here, we aim to evaluate the cytotoxic effects of curcumin on the expression of miR-383-5p and miR-708-5p and their target genes in CD44+ CSCs and CD44- non-CSCs isolated from the PC3 prostate cancer cell line. Materials and Methods: We used MTT assay to determine the optimal cytotoxic dose of curcumin on CD44± PC cells. Then, we assessed nuclear morphological changes using DAPi staining. We used Annexin V-FITC/PI to quantify apoptotic cell death. qRT-PCR was also used to detect miRNA and gene expression levels after curcumin treatment. Results: Curcumin significantly enhanced the apoptosis in both CD44- and CD44+ PC cells in a dose-dependent manner (p < 0.05). The cytotoxicity of curcumin against CD44- cells (IC50 40.30±2.32 µM) was found to be greater than that against CD44+ cells (IC50 83.31±2.91 µM). Also, curcumin promoted miR-383-5p and miR-708-5p overexpression while downregulating their target genes LDHA, PRDX3, and RAP1B, LSD1, respectively. Conclusion: Our findings indicate that curcumin, by promoting the expression of tumor suppressors, miR-383-5p and miR-708-5p, and inhibiting their target genes, induced its cytotoxicity against CD44± PC cells. We trust that curcumin could be established as a promising adjuvant therapy to current PC treatment options following more research in clinical settings.

Curcumin Upregulates miR-148a to Increase the Chemosensitivity of CD44-Positive Prostate Cancer Stem Cells to Paclitaxel Through Targeting the MSK1/IRS1 axis.

BACKGROUND: In men, prostate cancer (PC) is the second most common cause of cancer-related death. However, paclitaxel resistance is a major challenge in advanced PC. Curcumin, a natural antioxidant, has been demonstrated to have cytotoxic effects on cancer stem cells (CSCs). The goal of this study is to explore if curcumin can help lower chemoresistance to paclitaxel through the regulation of miR-148a-mediated apoptosis in prostate CSCs. METHODS: The 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and 4',6-diamidino-2-phenylindole (DAPi) labeling were used to determine cell survival. Immunohistochemistry was used to detect the expression of P-glycoprotein protein (P-gp) and CD44 proteins. Finally, real-time PCR was used to evaluate the regulatory effects of curcumin and paclitaxel on miR-148a and its target genes. RESULTS: Curcumin and paclitaxel co-treatment significantly reduced the IC50 value in CD44+cells compared to paclitaxel alone. Additionally, combining these drugs considerably increased apoptosis in CD44+cells. We also discovered that when curcumin and paclitaxel were combined, the expression of CD44 and P-gp was significantly reduced compared to paclitaxel alone. Curcumin and paclitaxel co-treatment also increased miR-148a levels and regulated the levels of its target genes MSK1 and IRS1. CONCLUSION: Curcumin may restore paclitaxel sensitivity by raising miR-148a expression and inhibiting its target genes.

Allicin Reduces 5-fluorouracil-resistance in Gastric Cancer Cells through Modulating MDR1, DKK1, and WNT5A Expression.

BACKGROUND & OBJECTIVE: 5-fluorouracil (5-FU) is approved for the treatment of gastric carcinoma (GC), but chemo-resistance limits the application of it for GC. Thus, the combination of 5-FU with adjuvants such as allicin may overcome multidrug resistance (MDR). METHODS: The anticancer effects of allicin, 5-FU, and allicin/5-FU on the 5-FU resistant MKN-45 cells were evaluated by MTT assay and DAPi staining. The expression of the P-glycoprotein (P-gp) and CD44 protein were determined using immunocytochemistry. We also quantified mRNA expression levels of WNT5A, Dickkopf-1 (DKK1), and MDR1 in the GC cells. RESULTS: Here, we found that the combination of allicin with 5-FU significantly increased apoptosis compared to 5-FU alone (P<0.05). We showed that WNT5A, MDR1, and DKK1 mRNA expression levels were down-regulated in the allicin- and allicin/5-FU-treated cells. Indeed, the combination of allicin and 5-FU significantly decreased the expression of the P-gp and CD44 proteins (P<0.05). CONCLUSION: Our findings indicate that the combination of allicin with 5-FU could reverse multidrug resistance in the GC cells by reducing the expression of WNT5A, DKK1, MDR1, P-gp, and CD44 levels.

Poly (L-lactic acid) nanofibrous scaffolds support the proliferation and neural differentiation of mouse neural stem and progenitor cells.

BACKGROUND: The distribution and growth of cells on nanofibrous scaffolds seem to be an indispensable precondition in cell tissue engineering. The potential use of biomaterial scaffolds in neural stem cell therapy is increasingly attracting attention. AIM: In this study, we produced porous nanofibrous scaffolds fabricated from random poly-L-lactic acid (PLLA) to support neurogenic differentiation of neural stem and progenitor cells (NSPCs), isolated from the subventricular zone (SVZ) of the adult mouse brain. METHODS: The viability and proliferation of the NSPCs on the nanofibrous PLLA scaffold were also tested by nuclear staining with 4, 6-diamidino-2-phenylindole dihydrochloride (DAPI), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and scanning electron microscopy (SEM). To investigate the differentiation potential of NSPCs on the scaffolds, the cells were treated with a neurogenic differentiation medium, and immunostaining was done to detect neuronal and glial cells after 14 and 21 days of cultivation. Furthermore, the morphology of differentiated cells on the scaffold was examined using SEM. RESULTS: The DAPI staining revealed the proliferation of NSPCs onto the surface of the nanofibrous PLLA scaffold. DAPI-positive cells were counted on days 2 and 5 after cultivation. The mean number of cells in each microscopic field was significantly (p < .05) increased (51 ± 19 on day 2 compared to 77 ± 25 cells on day 5). The results showed that the cell viability on PLLA scaffolds significantly increased compared to control groups. Moreover, cell viability was significantly increased 5 days after culturing (262.3 ± 50.2) as compared to 2 days culture in Vitro (174.2 ± 28.3, p < .05). Scanning electron micrographs also showed that the NSPCs adhered and differentiated on PLLA scaffolds. We found that the neural cell markers, microtubule-associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP), were expressed in NSPCs seeded on random PLLA scaffolds after 21 days of cultivation. CONCLUSION: These results suggest that the PLLA nano-scaffolds, due to their biocompatible property, are an appropriate structure for the proliferation, differentiation, and normal growth of NSPCs.

Thiamine reduced metabolic syndrome symptoms in rats via down-regulation of hepatic nuclear factor-kß and induction activity of glyoxalase-I.

OBJECTIVES: Metabolic syndrome (MS) is a cause of death worldwide. The hepatic nuclear factor- NF-kß (NF-kß) is the cardinal player of hepatic homeostasis, insulin sensitivity, and lipid metabolism. Thus, we investigated the effect of thiamine on hepatic gene expression of NF-kß and its levels of activators in MS rats. MATERIALS AND METHODS: Male Wistar rats were randomly divided into 4 equal groups (ten rats in each group): normal, MS, and two alike groups under thiamine treatment. MS was induced in rats with a high sucrose solution (40 % in drinking water) for 4 months. Treated groups of rats received 0.18 % of thiamine daily in drinking water. Hematoxylin-Eosin stains were employed to determine the histopathological changes of the liver. Metabolic profile, glycation products, oxidative stress, inflammatory markers, the activity of glyoxalase-I, as well as NF-kß hepatic expression of all rat groups, were determined. RESULTS: Acute hepatitis was not observed in the livers of the thiamine treated MS rats. Besides, the treatment showed an advantageous effect on glucose, lipid metabolism, and body weight via down-regulation of hepatic NF-kß and induction of glyoxalase system activity. Furthermore, the treatment decreased diverse glycation, oxidative stress, and inflammatory markers (P>0.001). CONCLUSION: Thiamine decreased body weight and improved metabolism and activity of glyoxalase-I in MS rats with anti-glycation, antioxidant, and anti-inflammatory activities. Further, the treatment had a hepato-protective effect via reduction of NF-kß signaling.

Efficacy of Metformin and Chemotherapeutic Agents on the Inhibition of Colony Formation and Shh/Gli1 Pathway: Metformin/Docetaxel Versus Metformin/5-Fluorouracil.

BACKGROUND: Gastric cancer is a common gastrointestinal cancer characterized by poor prognosis and chemoresistance. Docetaxel and 5-fluorouracil (5-FU) are frequently used for the treatment of gastric cancer. Despite their potent anti-cancer effects, chemoresistance occurs in metastatic gastric cancer. Metformin, a popular anti-diabetic drug, has been proven to have potent anticancer effects on gastrointestinal cancers. Here, we aim to improve this chemotherapy agents' efficacy by pretreatment with metformin. METHODS: The AGS gastric cancer cell line were pretreated with three different sub-toxic concentration of metformin and then treated with various concentrations of 5-FU and docetaxel.The anticancer effects of the combination of metformin with the chemotherapy agents were determined using clonogenic assay and DAPi staining. We used real-time PCR to evaluate Gli1, Gli2, and TWIST1 mRNA expression levels in the gastric cancer cells. Also, the expression of the Shh protein was assessed using immunocytochemistry. RESULTS: Here, we found that metformin sensitized the gastric cancer cells to chemotherapy. The combination treatments were more effective in reducing the number of cancer colonies compared to 5-FU or docetaxel alone. The combination of metformin with 5-FU or docetaxel significantly reduced the number of cells expressing the Shh protein compared to the 5-FU alone or docetaxel alone. Interestingly, we found that the combination of metformin with docetaxel significantly down-regulated the mRNA levels of Gli1, Gli2, and TWIST1 in the AGS gastric cancer cell line compared to docetaxel alone. CONCLUSION: Overall, our data strongly support an important role for metformin as an enhancer of the efficacy of chemotherapeutic agents against gastric cancer.

In Vitro Anticancer Effects of All-trans Retinoic Acid in Combination with Dacarbazine against CD117+ Melanoma Cells.

BACKGROUND: Malignant melanoma is a common form of skin cancer that contains different cell types recognized by various cell surface markers. Dacarbazine-based combination chemotherapy is frequently used for the treatment of melanoma. Despite its potent anticancer properties, resistance to dacarbazine develops in malignant melanoma. Here, we aim to improve response to dacarbazine therapy by pretreatment with all-trans retinoic acid (ATRA) in CD117+ melanoma cells. METHODS: The CD117+ melanoma cells were sorted from A375 malignant melanoma cell line using magnetic-activated cell sorting (MACS). The cell viability was examined by cell proliferation assay (MTT). Apoptosis was determined by acridine orange/ ethidium bromide staining. Indeed, we performed flow cytometry to evaluate the cell cycle arrest. RESULTS: Here, the CD117+ melanoma cells were incubated with various concentrations of ATRA, dacarbazine, and their combination to determine IC50 values. We found that 20 µM ATRA treatment followed by dacarbazine was found to be more effective than dacarbazine alone. There was an indication that the combination of ATRA with dacarbazine (ATRA/dacarbazine) caused more apoptosis and necrosis in the melanoma cells (P<0.05). Furthermore, ATRA/dacarbazine treatment inhibited the cell at the G0/G1 phase, while dacarbazine alone inhibited the cells at S phase. CONCLUSION: Collectively, combined treatment with ATRA and dacarbazine induced more apoptosis and enhanced the cell cycle arrest of CD117+ melanoma cells. These results suggested that ATRA increased the sensitivity of melanoma cells to the effect of dacarbazine.

Azelaic acid stimulates catalase activation and promotes hair growth through upregulation of Gli1 and Gli2 mRNA and Shh protein.

OBJECTIVE: Although azelaic acid is effective for treatment of acne and rosacea, the biological activity of azelaic acid and the effect of its combination therapy with minoxidil were not elucidated with regard to hair growth. MATERIALS AND METHODS: In this study, mouse vibrissae follicles were dissected on day 10 after depilation. Then, the bulb and bulge cells of the hair follicle were treated with minoxidil and azelaic acid for 10 days to evaluate Sonic hedgehog (Shh) protein expression. Moreover, bulge and bulb cells of the hair follicles were cultivated and the expression of Gli1, Gli2, and Axin2 mRNA levels was evaluated using real-time polymerase chain reaction (PCR) analysis. We further investigated the protective effects of azelaic acid against ultraviolet B (UVB) irradiation in cultured bulb and bulge cells by determining catalase activity. An irradiation dose of 20 mJ/cm2 UVB for 4 sec was chosen. RESULTS: The results showed that catalase activity significantly (p<0.05) increased in the bulge cells after exposure to 2.5 mM and 25 mM azelaic acid. Meanwhile, treatment of the bulb cells with azelaic acid (2.5 and 25 mM) did not cause significant changes in catalase activity. We also found that azelaic acid (25 mM) alone upregulated Gli1 and Gli2 expression in the bulge cells and 100 µ minoxidil caused Gli1 and Axin2 overexpression in the bulb region of the hair follicle. Moreover, minoxidil (100 µM) alone and in combination with azelaic acid (25 mM) led to Shh protein overexpression in the hair follicles in vitro and in organ culture. CONCLUSION: Our results indicated a potential role for azelaic acid in the protection of bulge cells from UVB damage and its combination with minoxidil may activate hair growth through overexpression of Shh protein.

Reduced Levels of miR-28 and miR-200a Act as Predictor Biomarkers of Aggressive Clinicopathological Characteristics in Gastric Cancer Patients.

BACKGROUND: MicroRNAs (miRNAs) play critical roles in different pathological processes including cancer development and progression. To find novel molecular diagnostic and prognostic markers and promising therapeutic tools for gastric cancer (GC), we aimed to investigate the relationship of the expression levels of miR-28-5p or miR-200a-3p with the clinicopathological criteria and to explore their impacts on the progression of human GC. MATERIALS AND METHODS: Quantitative RT-PCR was performed to analyze miR-28 and miR-200a expression in 60 GC and 60 non-GC tissue samples. RESULT: Our results revealed that the expressions of miR-200a and miR-28 were significantly downregulated in GC in comparison with non- GC tissues. Tumors with low miR-28 expression had larger tumor size, more advanced histological grade, and a higher incidence of lymph node and distal metastasis than the tumors with high miR-28 expressions. Furthermore, receiver operating characteristic (ROC) analyses demonstrate that the expression of miR-28 is a predictive biomarker allows predicting the histological grade, tumor size, and occurrence of nodal and distal metastases. We also found a significant inverse association between miR-200a expression and the rate of lymph node metastasis (p = 0.010, r = -0.334). CONCLUSION: Our findings suggest that the miR-28 and miR-200a have tumor-suppressor functions and may be considered as potential biomarkers for gastric cancer diagnosis and prognosis.

miR-124 promotes neural differentiation in mouse bulge stem cells by repressing Ptbp1 and Sox9.

Hair follicle stem cells (HFSCs) are able to differentiate into neurons and glial cells. Distinct microRNAs (miRNAs) regulate the proliferation and differentiation of HFSCs. However, the exact role of miR-124 in the neural differentiation of HFSCs has not been elucidated. HFSCs were isolated from mouse whisker follicles. miR-9, let-7b, and miR-124, Ptbp1 , and Sox9 expression levels were detected by real-time polymerase chain reaction (RT-PCR). The influence of miR-124 transfection was evaluated using immunostaining. We demonstrated that miR-124 and let-7b expression levels were significantly increased after the neural differentiation. Sox9 and Ptbp1 were identified as the target of miR-124 in the HFSCs. During neural differentiation and miR-124 mimicking, Ptbp1 and Sox9 levels were decreased. Moreover, the miR-124 overexpression increased MAP2 (58.43 ± 11.26) and NeuN (48.34 ± 11.15) proteins expression. The results demonstrated that miR-124 may promote the differentiation of HFSCs into neuronal cells by targeting Sox9 and Ptbp1.

Molecular mechanism and cytotoxicity of allicin and all-trans retinoic acid against CD44+ versus CD117+ melanoma cells.

BACKGROUND: All-trans retinoic acid (ATRA) is a differentiating agent that inhibits cancer cell growth during the cell cycle. However, despite its potent antitumor properties, some melanoma cells are resistant to ATRA therapy. PURPOSE: Here, we hypothesized that allicin can sensitize malignant melanoma cells to ATRA treatment. To clarify this mechanism, we determined the sensitivity to ATRA, allicin and allicin/ATRA in CD44+ and CD117+ melanoma cell subpopulations. METHODS: The CD44+and CD117+cells were sorted from A375 melanoma cell line using the magnetic-activated cell sorting (MACS). The potential anticancer effects of ATRA, allicin and allicin/ATRA were examined using cell proliferation MTT assay. In addition, flow cytometry was used to detect cell cycle arrest. The efficacy of the treatments in controlling cancer cell proliferation was assessed by quantitative realtime polymerase chain reaction (RT-PCR). RESULTS: Here, we demonstrated that CD44+ melanoma cells were more resistant to allicin and ATRA than CD117+ cells. Importantly, we observed that allicin sensitized melanoma cell to ATRA-induced cell death. The combination treatment with allicin and ATRA significantly reduced the IC50 value obtained for ATRA alone in CD44+ melanoma cells. In CD44+ cells, the IC50 value of ATRA was 37.43 ±â€¯0.54, while the IC50 value of allicin/ATRA treatment was 17.53 ±â€¯0.2 µM. Allicin treatment resulted in significant increases in the percentage of cells at the G2/M and G0/G1 phases in the CD44+ and CD117+ cells, respectively. The combination treatment caused the inhibition of CD44+ and CD117+ melanoma cells at the S phases compared to ATRA alone. Allicin, ATRA, and allicin/ATRA increased the expression of cyclin D1 mRNA in both CD44+ and CD117+ cells. Allicin combination with ATRA increased the mRNA level of RARß in CD117+ cells. Furthermore, allicin alone caused a remarkable reduction of MMP-9 mRNA expression in both CD44+ and CD117+ cells. In contrast, ATRA and the combination treatment significantly increased MMP-9 gene expression in CD44+ cells. CONCLUSION: Overall, our results indicate that allicin reinforces the ATRA-mediated inhibitory effects on CD44+ and CD117+ melanoma cells and may provide a new approach for the treatment of malignant melanoma.

Epi/perineural and Schwann Cells as Well as Perineural Sheath Integrity are Affected Following 2,4-D Exposure.

2,4-dicholorophenoxy acetic acid (2,4-D) is a worldwide-known hormone herbicide. However, there are increasing concerns about its exposure and risks of developing pathological conditions for the peripheral nervous system. The aim of this study was to investigate the mechanism(s) involved in the toxicity of 2,4-D on peripheral nerve's cellular components. The epi/perineural and Schwann cells and a total of three cell lines were treated with 2,4-D. The viability of cells at different doses of 2,4-D was measured by MTT assay. The cell cycle analyses, cumulative cell counting, fluorescent staining, antioxidant and caspase enzymes activity were examined on epi/perineural and Schwann cells. The epi/perineural cells were assessed as having biological macromolecular changes. Some tight junction-related genes and proteins were also tested on explants of 2,4-D treated epi/perineural tissue. The viability of 2,4-D treated cells was reduced in a dose-dependent manner. Reduced growth rate and G1 cell cycle arrest were verified in 2,4-D treated epi/perineural and Schwann cells. The use of staining methods (acridine orange/ethidium bromide and DAPI) and caspase 3/7 activity assay along with malondialdehyde, glutathione peroxidase, and superoxide dismutase activity assays indicated the apoptotic and oxidant effects of 2,4-D on epi/perineural and Schwann cells. Data obtained from FTIR revealed changes in epi/perineural proteins and cell membrane lipids. Additionally, claudin-1, occludin, and ZO-1 gene/protein expression profiles were significantly reduced in 2,4-D-treated epi/perineural pieces. Our data indicated that oxidative stress, apoptosis of epi/perineural and Schwann cell and impaired blood-nerve barrier may have contributed to nerve damage following 2,4-D exposure.

Human platelet lysate versus minoxidil stimulates hair growth by activating anagen promoting signaling pathways.

Minoxidil and human platelet lysate (HPL) are commonly used to treat patients with hair loss. However, the roles of HPL versus minoxidil in hair follicle biology largely remain unknown. Here, we hypothesized that bulge and dermal papilla (DP) cells may express specific genes, including Kras, Erk, Akt, Shh and ß-catenin after exposure to minoxidil or HPL. The mouse hair follicles were isolated on day 10 after depilation and bulge or DP regions were dissected. The bulge and DP cells were cultured for 14days in DMEM/F12 medium. Then, the cells were treated with 100µM minoxidil and 10% HPL for 10 days. Nuclear morphology was identified using DAPi staining. Reverse transcriptase and real-time polymerase chain reaction (PCR) analysis were also performed to examine the expression of Kras, Erk, Akt, Shh and ß-catenin mRNA levels in the treated bulge and DP regions after organ culture. Here, we found that minoxidil influences bulge and DP cell survival (P<0.05). Apoptosis in DP cells was also meaningfully decreased by HPL treatment (P=0.014). In addition, Kras, Akt, Erk, Shh and ß-catenin mRNA levels were changed in response to minoxidil treatment in both bulge and DP cells. HPL mediated Erk upregulation in both bulge and DP cells (P<0.05), but Kras and Akt mRNA levels were not considerably different in the HPL-treated cells. ß-catenin mRNA level was also significantly increased in the bulge region by HPL. We also found that Shh mRNA level was considerably higher in HPL-treated bulge cells than in minoxidil-treated bulge cells. In contrast, the expression of ß-cateinin and Shh in the DP cells was not meaningfully increased after treatment with HPL. Our results suggest that minoxidil and HPL can promote hair growth by activating the main anagen inducing signaling pathways.

Highly Efficient Neural Differentiation of CD34-Positive Hair-Follicle-Associated Pluripotent Stem Cells Induced by Retinoic Acid and Serum-Free Medium.

Neural differentiation of hair-follicle-associated pluripotent (HAP) stem cells residing in the bulge area is a promising autologous source for stem cell therapy. In the present chapter, we describe the identification and enrichment of CD34(+) HAP stem cells by magnetic-activated cell sorting (MACS), and induce them to differentiate into neuronal and glial cells using defined neural-induction media. The different neural cell populations arising during in vitro differentiation from HAP stem cells are characterized by reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry assay.

Low-dose all-trans retinoic acid enhances cytotoxicity of cisplatin and 5-fluorouracil on CD44(+) cancer stem cells.

Cis-diamminedichloridoplatinum(II)(CDDP)-based combination chemotherapy is frequently used in gastrointestinal cancer. The synergistic mechanism of all-trans retinoic acid (ATRA), cisplatin (CDDP) and 5-fluorouracil (5-FU) in combination remains unclear. Despite their potent antitumor properties, resistance to CDDP and 5-FU develops frequently in tumors. To clarify this mechanism, we determined the sensitivity to each drug and their combination in two gastrointestinal cancer stem cells (CSCs) subpopulation. Here, we report the identification and separation of CD44(+) cells from human gastric carcinoma (AGS) and human esophageal squamous cell carcinoma (KYSE-30) cancer cell lines by magnetic activated cell sorting (MACS). We allowed the CD44(±) cells to grow 6 days at a subtoxic concentration of ATRA and then treated with different concentration of CDDP and 5-FU for 24h. The cytotoxicity was examined by cell proliferation MTT assay. Additionally, AO/EB staining was used for detection of apoptotic cells. In order to determine whether the growth inhibition was also associated with changes in cell cycle distribution, cell cycle analysis was performed using flow cytometry. Low concentration of ATRA (1µM, 6days) followed by 5-FU and CDDP was found to be more effective than either drugs alone, thus resulting in synergistic cytotoxicity in Kyse-30 and AGSCD44(±) cells. Furthermore, there was an indication that the combination of ATRA with 5FU and CDDP caused an increase in cell cycle arrest in G2/M and G0/G1. We conclude that low concentration of ATRA enhances the cytotoxicity of CDDP and 5FU by facilitating apoptosis and cell cycle arrest in gastrointestinal CSCs and provide a rational basis for the design of novel, well-tolerated CDDP- and 5FU-based chemotherapy in human gastrointestinal carcinoma.

Hair follicle stem cells: In vitro and in vivo neural differentiation.

Hair follicle stem cells (HFSCs) normally give rise to keratinocytes, sebocytes, and transient amplifying progenitor cells. Along with the capacity to proliferate rapidly, HFSCs provide the basis for establishing a putative source of stem cells for cell therapy. HFSCs are multipotent stem cells originating from the bulge area. The importance of these cells arises from two important characteristics, distinguishing them from all other adult stem cells. First, they are accessible and proliferate for long periods. Second, they are multipotent, possessing the ability to differentiate into mesodermal and ectodermal cell types. In addition to a developmental capacity in vitro, HFSCs display an ability to form differentiated cells in vivo. During the last two decades, numerous studies have led to the development of an appropriate culture condition for producing various cell lineages from HFSCs. Therefore, these stem cells are considered as a novel source for cell therapy of a broad spectrum of neurodegenerative disorders. This review presents the current status of human, rat, and mouse HFSCs from both the cellular and molecular biology and cell therapy perspectives. The first section of this review highlights the importance of HFSCs and in vitro differentiation, while the final section emphasizes the significance of cell differentiation in vivo.

In vitro neural differentiation of CD34 (+) stem cell populations in hair follicles by three different neural induction protocols.

Differentiation of hair follicle stem cells (HFSCs) into neurons and glial cells represents a promising cell-based therapy for neurodegenerative diseases. The hair follicle bulge area is reported as a putative source of new stem cell population for many years. In vitro studies have implicated neural differentiation of HFSCs. Here, we report the identification and purification of CD34 (+) cells from hair follicle by magnetic activated cell sorting (MACS). We next determined the cytotoxic effects of all-trans retinoic acid (RA) by using cell viability assays. Moreover, the neural differentiation potential of CD34 (+) cells was evaluated in the presence of RA, serum-free condition, and neural differentiation medium (NDM) treatments by using immunocytochemistry and reverse transcription polymerase chain reaction (RT-PCR). Our results showed that the isolated CD34 (+) stem cells were 12% of the total cells in the bulge area, and the neural cells derived from the stem cells expressed nestin, microtubule-associated protein 2 (MAP2), and glial fibrillary acidic protein (GFAP). Interestingly, all the neural induction media supported neuronal differentiation most effectively, but treatment with serum-free medium significantly increased the number of GFAP-positive glial cells. Moreover, increasing RA concentration (≥10 µM) leads to increased cell death in the cells, but a lower concentration of RA (1 µM) treatment results in a decrease in CD34-expressing stem cells. These findings show an instructive neuronal effect of three neural induction media in HFSCs, indicating the important role of this induction media in the specification of the stem cells toward a neural phenotype.

Cytotoxic Effects of Newly Synthesized Palladium(II) Complexes of Diethyldithiocarbamate on Gastrointestinal Cancer Cell Lines.

As a part of a drug development program to discover novel therapeutic and more effective palladium (Pd) based anticancer drugs, a series of water-soluble Pd complexes have been synthesized by interaction between [Pd (phen)(H2O)2(NO3)2] and alkylenebisdithiocarbamate(al-bis-dtc) disodium salts. This study was undertaken to examine the possible cytotoxic effect of three novel complexes (0.125-64 µg/mL) on human gastric carcinoma (AGS), esophageal squamous cell carcinoma (Kyse-30), and hepatocellular carcinoma (HepG2) cell lines. The cytotoxicity was examined using cell proliferation and acridine orange/ethidium bromide (AO/EB) assay. In order to examine the effects of new Pd(II) complexes on cell cycle status, we performed cell cycle analysis. The complexes were found to have completely lethal effects on the cell lines, and the half maximal inhibitory concentration (IC50) values obtained for the cell lines were much lower in comparison with cisplatin. We demonstrated that the three new Pd(II) complexes are able to induce G2/M phase arrest in AGS and HepG2; in addition, the Pd(II) complexes caused an S phase arrest in Kyse-30 cell line. Our results indicate that newly synthesized Pd(II) complexes may provide a novel class of chemopreventive compounds for anticancer therapy.

Rat hair follicle stem cells differentiate and promote recovery following spinal cord injury.

Emerging studies of treating spinal cord injury (SCI) with adult stem cells led us to evaluate the effects of transplantation of hair follicle stem cells in rats with a compression-induced spinal cord lesion. Here, we proposed a hypothesis that rat hair follicle stem cell transplantation can promote the recovery of injured spinal cord. Compression-induced spinal cord injury was induced in Wistar rats in this study. The bulge area of the rat vibrissa follicles was isolated, cultivated and characterized with nestin as a stem cell marker. 5-Bromo-2'-deoxyuridine (BrdU) labeled bulge stem cells were transplanted into rats with spinal cord injury. Immunohistochemical staining results showed that some of the grafted cells could survive and differentiate into oligodendrocytes (receptor-interacting protein positive cells) and neuronal-like cells (ßIII-tubulin positive cells) at 3 weeks after transplantation. In addition, recovery of hind limb locomotor function in spinal cord injury rats at 8 weeks following cell transplantation was assessed using the Basso, Beattie and Bresnahan (BBB) locomotor rating scale. The results demonstrate that the grafted hair follicle stem cells can survive for a long time period in vivo and differentiate into neuronal- and glial-like cells. These results suggest that hair follicle stem cells can promote the recovery of spinal cord injury.

Effects of tamoxifen on morphological and ultrastructural aspects of developing hippocampus of rat.

BACKGROUND: Tamoxifen treatment induced cell death in the hippocampus formation of the prenatal and postnatal rat. The present study delineates the effect of tamoxifen on developing hippocampus in prenatal, postnatal and full term neonate rats received certain doses of the partial antagonist tamoxifen. METHODS: After perfusion and fixation, the brains were removed and processed for light and electron microscopy. The morphology, ultrastructure and the density of the neurons in different ages (E22, P1, P7 and P21) and in different areas of developing hippocampus including cornu ammonis (CA1 and CA3), dentate gyrus and subiculum were studied. RESULTS: These findings showed that in tamoxifen-treated groups, the cell number of pyramidal neurons of CA1 and subiculum significantly decreased comparing to control groups in E22, P1 and P7 but not in third weeks. The mitochondria of the above mentioned groups also showed a dilated feature with less cristae than control group and most of them were greatly enlarged and swollen into spherical shapes rather than the normal ovoid or rod shape. CONCLUSION: The present study shows that prenatal exposure to tamoxifen alters neurogenesis in developing rat hippocampus. These results demonstrated the non-neuroprotective roles of tamoxifen.