<p>Cells were lentivirally transfected with Hsp27 shRNA (shR1 and shR2) or scrambled shRNA,... more <p>Cells were lentivirally transfected with Hsp27 shRNA (shR1 and shR2) or scrambled shRNA, then exposed to hypoxia and serum depletion. CD133+ and CD133− cells were isolated using MACS separation 4 days later. (A) Immunoblot analysis for HT-29 protein levels of CD133+ and CD133− cells after MACS separation. (B) The percentage of CD133+ cells were analyzed by flow cytometry and the increased CD133+ percentage compared to CD133+ percentage in normoxia and growth medium was calculated. (C) Cells were re-exposed to hypoxia and serum depletion for 1 day, followed by TUNEL staining. (D-F) HT-29 cells were exposed to serum depletion and hypoxia in the presence of Quercetin, KRIBB3, or DMSO (vehicle control) for 4 days. (D) CD133+ cell were isolated and the protein levels were analyzed by western blotting. (E) The increased percentage of CD133+ cells and (F) apoptosis of CD133+ cells were analyzed by flow cytometry and TUNEL staining, respectively. Error bars represent standard deviations. (**p<0.01 compared with the scrambled as determined by the Student’s t test.)</p
<p>(A) HT-29, (B) CCS and HCW cells were seeded with normal growth medium (FBS) under normo... more <p>(A) HT-29, (B) CCS and HCW cells were seeded with normal growth medium (FBS) under normoxia (Nor). Cells were exposed to serum depletion (SF), hypoxia (Hyp), hypoxia and serum depletion, or, as a control, under the same condition, and 2, 4 or 6 days later, the cells were harvested for assaying CD133 percentage by flow cytometry. (C left panel) Total cell numbers for each condition and (C right panel) cell number of CD133− and CD133+ of HT-29 cells at indicated period under hypoxia and serum depletion were measured. (D left panel) Total cell number and (D right panel) numbers of CD133+ and CD133− cells of CCS and HCW cells at 4 days under each condition. (E and F) Flow cytometry for surface protein profiles of cells cultured under control, or under hypoxia and serum depletion for 4 days.</p
<p>CCS and HCW colorectal cancer cells, A549 lung cancer cells, HTB186 medulloblastoma cell... more <p>CCS and HCW colorectal cancer cells, A549 lung cancer cells, HTB186 medulloblastoma cells and SAS, oral cancer cells were continually cultured under serum depletion in the presence of EGF (10 ng/mL) and FGF2 (10 ng/mL) to enrich TICs (E+F) or in serum-containing medium (CTR) as a control. (A) Immunoblots for pluripotent markers. (B) Immunoblots for phosphorylated PP2A (pPP2A), PP2A and Hsp27. (C) Immunoblots for caspase 9 and 3. (D) Schema demonstrates the antiapoptosis pathway of colorectal TICs in response to in vitro hypoxia and serum depletion.</p
Although oncolytic viruses are currently being evaluated for cancer treatment in clinical trials,... more Although oncolytic viruses are currently being evaluated for cancer treatment in clinical trials, systemic administration is hindered by many factors that prevent them from reaching the tumor cells. When administered systemically, mesenchymal stem cells (MSCs) target tumors, and therefore constitute good cell carriers for oncolytic viruses. MSCs were primed with trichostatin A under hypoxia, which upregulated the expression of CXCR4, a chemokine receptor involved in tumor tropism, and coxsackievirus and adenovirus receptor that plays an important role in adenoviral infection. After priming, MSCs were loaded with conditionally replicative adenovirus that exhibits limited proliferation in cells with a functional p53 pathway and encodes Escherichia coli nitroreductase (NTR) enzymes (CRAdNTR) for targeting tumor cells. Primed MSCs increased tumor tropism and susceptibility to adenoviral infection, and successfully protected CRAdNTR from neutralization by anti-adenovirus antibodies both ...
<p>(A) TUNEL staining for apoptosis was performed after MACS separation of CD133− and CD133... more <p>(A) TUNEL staining for apoptosis was performed after MACS separation of CD133− and CD133+ cells under hypoxia and serum depletion for 4 days. (B) CD133− and CD133+ cells under hypoxia and serum depletion for 3 days were harvested by MACS separation, reseeded under hypoxia and serum depletion, and APOPercentage assay was performed 16 h later. (C) Quantitative RT-PCR for mRNA levels. (D-F) Cell lysates of CD133+ and CD133− cells after MACS separation at day 4 of exposure to hypoxia and serum depletion were prepared and used for (D, F) immunoblot analysis for protein levels, and (E) MAPK phospho-antibody array for analyzing the levels of indicated signaling pathways. Graphs show each signaling after normalized with control. CD133+ cells decreased in the expression and the activation of caspase 9 and 3 and increased in the expression of phospho-Hsp27. Error bars represent standard deviations. (*p<0.05 and **p<0.01 as determined by the Student’s t test.) Data of HT-29 cells are shown as representative results.</p
Fluorenyl-9-methoxycarbonyl (Fmoc)-diphenylalanine (Fmoc-FF) and Fmoc-arginine-glycine--aspartate... more Fluorenyl-9-methoxycarbonyl (Fmoc)-diphenylalanine (Fmoc-FF) and Fmoc-arginine-glycine--aspartate (Fmoc-RGD) peptides self-assemble to form a 3D network of supramolecular hydrogel (Fmoc-FF/Fmoc-RGD), which provides a nanofibrous network that uniquely presents bioactive ligands at the fiber surface for cell attachment. In the present study, mesenchymal stem cells (MSCs) in Fmoc-FF/Fmoc-RGD hydrogel increase in proliferation and survival compared to those in Fmoc-FF/Fmoc-RGE hydrogel. Moreover, MSCs encapsulated in Fmoc-FF/Fmoc-RGD hydrogel and induced in each defined induction medium undergo in vitro osteogenic, adipogenic, and chondrogenic differentiation. For in vivo differentiation, MSCs encapsulated in hydrogel are induced in each defined medium for one week, followed by injection into gelatin sponges and transplantation into immunodeficient mice for four weeks. MSCs in Fmoc-FF/Fmoc-RGD hydrogel increase in differentiation into osteogenic, adipogenic, and chondrogenic differentia...
Targeting tumour-initiating cells (TICs) would lead to new therapies to cure cancer. We previousl... more Targeting tumour-initiating cells (TICs) would lead to new therapies to cure cancer. We previously demonstrated that TICs have the capacity to survive under suspension conditions, while other cells undergo anoikis. Here we show that TICs exhibit increased phosphorylation levels of S727STAT3 because of PP2A inactivation. Collagen 17 gene expression is upregulated in a STAT3-dependent manner, which also stabilizes laminin 5 and engages cells to form hemidesmosome-like junctions in response. Blocking the PP2A-S727STAT3-collagen 17 pathway inhibits the suspension survival of TICs and their ability to form tumours in mice, while activation of the same pathway increases the suspension survival and tumour-initiation capacities of bulk cancer cells. The S727STAT3 phosphorylation levels correlate with collagen 17 expression in colon tumour samples, and correlate inversely with survival. Finally, this signalling axis enhances the ability of TIC to form tumours in mouse models of malignant lun...
Many cell therapies currently being tested are based on mesenchymal stromal cells (MSCs). However... more Many cell therapies currently being tested are based on mesenchymal stromal cells (MSCs). However, MSCs start to enter the senescent state upon long-term expansion. The role of retinoblastoma (RB) protein in regulating MSC properties is not well studied. Here, we show that RB levels are higher in early-passage MSCs compared with late-passage MSCs. RB knockdown induces premature senescence and reduced differentiation potentials in early-passage MSCs. RB overexpression inhibits senescence and increases differentiation potentials in late-passage MSCs. Expression of DNMT1, but not DNMT3A or DNMT3B, is also higher in early-passage MSCs than in late-passage MSCs. Furthermore, DNMT1 knockdown in early-passage MSCs induces senescence and reduces differentiation potentials, whereas DNMT1 overexpression in late-passage MSCs has the opposite effect. These results demonstrate that RB expressed in early-passage MSCs upregulates DNMT1 expression and inhibits senescence in MSCs. Therefore, genetic...
Antiangiogenesis is an efficient therapy for eliminating colon cancers, but because of recurrence... more Antiangiogenesis is an efficient therapy for eliminating colon cancers, but because of recurrence it remains only palliative. We hypothesized that certain populations of tumor cells resist antiangiogenesis-induced apoptosis and explored the underlying mechanism. We demonstrated that the CD133(+) population of cells in colon cancer is resistant to anti-angiogenesis therapy. Additionally, we identified an anti-apoptotic signaling pathway responsible for this resistance involving PP2A, p38MAPK, MAPKAPK2, and Hsp27. Thus, this pathway may offer a new avenue to develop target therapy for colorectal cancer.
Targeting tumour-initiating cells (TICs) would lead to new therapies to cure cancer. We previousl... more Targeting tumour-initiating cells (TICs) would lead to new therapies to cure cancer. We previously demonstrated that TICs have the capacity to survive under suspension conditions, while other cells undergo anoikis. Here we show that TICs exhibit increased phosphorylation levels of S727STAT3 because of PP2A inactivation. Collagen 17 gene expression is upregulated in a STAT3-dependent manner, which also stabilizes laminin 5 and engages cells to form hemidesmosome-like junctions in response. Blocking the PP2A-S727STAT3-collagen 17 pathway inhibits the suspension survival of TICs and their ability to form tumours in mice, while activation of the same pathway increases the suspension survival and tumour-initiation capacities of bulk cancer cells. The S727STAT3 phosphorylation levels correlate with collagen 17 expression in colon tumour samples, and correlate inversely with survival. Finally, this signalling axis enhances the ability of TIC to form tumours in mouse models of malignant lun...
<p>Cells were lentivirally transfected with Hsp27 shRNA (shR1 and shR2) or scrambled shRNA,... more <p>Cells were lentivirally transfected with Hsp27 shRNA (shR1 and shR2) or scrambled shRNA, then exposed to hypoxia and serum depletion. CD133+ and CD133− cells were isolated using MACS separation 4 days later. (A) Immunoblot analysis for HT-29 protein levels of CD133+ and CD133− cells after MACS separation. (B) The percentage of CD133+ cells were analyzed by flow cytometry and the increased CD133+ percentage compared to CD133+ percentage in normoxia and growth medium was calculated. (C) Cells were re-exposed to hypoxia and serum depletion for 1 day, followed by TUNEL staining. (D-F) HT-29 cells were exposed to serum depletion and hypoxia in the presence of Quercetin, KRIBB3, or DMSO (vehicle control) for 4 days. (D) CD133+ cell were isolated and the protein levels were analyzed by western blotting. (E) The increased percentage of CD133+ cells and (F) apoptosis of CD133+ cells were analyzed by flow cytometry and TUNEL staining, respectively. Error bars represent standard deviations. (**p<0.01 compared with the scrambled as determined by the Student’s t test.)</p
<p>(A) HT-29, (B) CCS and HCW cells were seeded with normal growth medium (FBS) under normo... more <p>(A) HT-29, (B) CCS and HCW cells were seeded with normal growth medium (FBS) under normoxia (Nor). Cells were exposed to serum depletion (SF), hypoxia (Hyp), hypoxia and serum depletion, or, as a control, under the same condition, and 2, 4 or 6 days later, the cells were harvested for assaying CD133 percentage by flow cytometry. (C left panel) Total cell numbers for each condition and (C right panel) cell number of CD133− and CD133+ of HT-29 cells at indicated period under hypoxia and serum depletion were measured. (D left panel) Total cell number and (D right panel) numbers of CD133+ and CD133− cells of CCS and HCW cells at 4 days under each condition. (E and F) Flow cytometry for surface protein profiles of cells cultured under control, or under hypoxia and serum depletion for 4 days.</p
<p>CCS and HCW colorectal cancer cells, A549 lung cancer cells, HTB186 medulloblastoma cell... more <p>CCS and HCW colorectal cancer cells, A549 lung cancer cells, HTB186 medulloblastoma cells and SAS, oral cancer cells were continually cultured under serum depletion in the presence of EGF (10 ng/mL) and FGF2 (10 ng/mL) to enrich TICs (E+F) or in serum-containing medium (CTR) as a control. (A) Immunoblots for pluripotent markers. (B) Immunoblots for phosphorylated PP2A (pPP2A), PP2A and Hsp27. (C) Immunoblots for caspase 9 and 3. (D) Schema demonstrates the antiapoptosis pathway of colorectal TICs in response to in vitro hypoxia and serum depletion.</p
Although oncolytic viruses are currently being evaluated for cancer treatment in clinical trials,... more Although oncolytic viruses are currently being evaluated for cancer treatment in clinical trials, systemic administration is hindered by many factors that prevent them from reaching the tumor cells. When administered systemically, mesenchymal stem cells (MSCs) target tumors, and therefore constitute good cell carriers for oncolytic viruses. MSCs were primed with trichostatin A under hypoxia, which upregulated the expression of CXCR4, a chemokine receptor involved in tumor tropism, and coxsackievirus and adenovirus receptor that plays an important role in adenoviral infection. After priming, MSCs were loaded with conditionally replicative adenovirus that exhibits limited proliferation in cells with a functional p53 pathway and encodes Escherichia coli nitroreductase (NTR) enzymes (CRAdNTR) for targeting tumor cells. Primed MSCs increased tumor tropism and susceptibility to adenoviral infection, and successfully protected CRAdNTR from neutralization by anti-adenovirus antibodies both ...
<p>(A) TUNEL staining for apoptosis was performed after MACS separation of CD133− and CD133... more <p>(A) TUNEL staining for apoptosis was performed after MACS separation of CD133− and CD133+ cells under hypoxia and serum depletion for 4 days. (B) CD133− and CD133+ cells under hypoxia and serum depletion for 3 days were harvested by MACS separation, reseeded under hypoxia and serum depletion, and APOPercentage assay was performed 16 h later. (C) Quantitative RT-PCR for mRNA levels. (D-F) Cell lysates of CD133+ and CD133− cells after MACS separation at day 4 of exposure to hypoxia and serum depletion were prepared and used for (D, F) immunoblot analysis for protein levels, and (E) MAPK phospho-antibody array for analyzing the levels of indicated signaling pathways. Graphs show each signaling after normalized with control. CD133+ cells decreased in the expression and the activation of caspase 9 and 3 and increased in the expression of phospho-Hsp27. Error bars represent standard deviations. (*p<0.05 and **p<0.01 as determined by the Student’s t test.) Data of HT-29 cells are shown as representative results.</p
Fluorenyl-9-methoxycarbonyl (Fmoc)-diphenylalanine (Fmoc-FF) and Fmoc-arginine-glycine--aspartate... more Fluorenyl-9-methoxycarbonyl (Fmoc)-diphenylalanine (Fmoc-FF) and Fmoc-arginine-glycine--aspartate (Fmoc-RGD) peptides self-assemble to form a 3D network of supramolecular hydrogel (Fmoc-FF/Fmoc-RGD), which provides a nanofibrous network that uniquely presents bioactive ligands at the fiber surface for cell attachment. In the present study, mesenchymal stem cells (MSCs) in Fmoc-FF/Fmoc-RGD hydrogel increase in proliferation and survival compared to those in Fmoc-FF/Fmoc-RGE hydrogel. Moreover, MSCs encapsulated in Fmoc-FF/Fmoc-RGD hydrogel and induced in each defined induction medium undergo in vitro osteogenic, adipogenic, and chondrogenic differentiation. For in vivo differentiation, MSCs encapsulated in hydrogel are induced in each defined medium for one week, followed by injection into gelatin sponges and transplantation into immunodeficient mice for four weeks. MSCs in Fmoc-FF/Fmoc-RGD hydrogel increase in differentiation into osteogenic, adipogenic, and chondrogenic differentia...
Targeting tumour-initiating cells (TICs) would lead to new therapies to cure cancer. We previousl... more Targeting tumour-initiating cells (TICs) would lead to new therapies to cure cancer. We previously demonstrated that TICs have the capacity to survive under suspension conditions, while other cells undergo anoikis. Here we show that TICs exhibit increased phosphorylation levels of S727STAT3 because of PP2A inactivation. Collagen 17 gene expression is upregulated in a STAT3-dependent manner, which also stabilizes laminin 5 and engages cells to form hemidesmosome-like junctions in response. Blocking the PP2A-S727STAT3-collagen 17 pathway inhibits the suspension survival of TICs and their ability to form tumours in mice, while activation of the same pathway increases the suspension survival and tumour-initiation capacities of bulk cancer cells. The S727STAT3 phosphorylation levels correlate with collagen 17 expression in colon tumour samples, and correlate inversely with survival. Finally, this signalling axis enhances the ability of TIC to form tumours in mouse models of malignant lun...
Many cell therapies currently being tested are based on mesenchymal stromal cells (MSCs). However... more Many cell therapies currently being tested are based on mesenchymal stromal cells (MSCs). However, MSCs start to enter the senescent state upon long-term expansion. The role of retinoblastoma (RB) protein in regulating MSC properties is not well studied. Here, we show that RB levels are higher in early-passage MSCs compared with late-passage MSCs. RB knockdown induces premature senescence and reduced differentiation potentials in early-passage MSCs. RB overexpression inhibits senescence and increases differentiation potentials in late-passage MSCs. Expression of DNMT1, but not DNMT3A or DNMT3B, is also higher in early-passage MSCs than in late-passage MSCs. Furthermore, DNMT1 knockdown in early-passage MSCs induces senescence and reduces differentiation potentials, whereas DNMT1 overexpression in late-passage MSCs has the opposite effect. These results demonstrate that RB expressed in early-passage MSCs upregulates DNMT1 expression and inhibits senescence in MSCs. Therefore, genetic...
Antiangiogenesis is an efficient therapy for eliminating colon cancers, but because of recurrence... more Antiangiogenesis is an efficient therapy for eliminating colon cancers, but because of recurrence it remains only palliative. We hypothesized that certain populations of tumor cells resist antiangiogenesis-induced apoptosis and explored the underlying mechanism. We demonstrated that the CD133(+) population of cells in colon cancer is resistant to anti-angiogenesis therapy. Additionally, we identified an anti-apoptotic signaling pathway responsible for this resistance involving PP2A, p38MAPK, MAPKAPK2, and Hsp27. Thus, this pathway may offer a new avenue to develop target therapy for colorectal cancer.
Targeting tumour-initiating cells (TICs) would lead to new therapies to cure cancer. We previousl... more Targeting tumour-initiating cells (TICs) would lead to new therapies to cure cancer. We previously demonstrated that TICs have the capacity to survive under suspension conditions, while other cells undergo anoikis. Here we show that TICs exhibit increased phosphorylation levels of S727STAT3 because of PP2A inactivation. Collagen 17 gene expression is upregulated in a STAT3-dependent manner, which also stabilizes laminin 5 and engages cells to form hemidesmosome-like junctions in response. Blocking the PP2A-S727STAT3-collagen 17 pathway inhibits the suspension survival of TICs and their ability to form tumours in mice, while activation of the same pathway increases the suspension survival and tumour-initiation capacities of bulk cancer cells. The S727STAT3 phosphorylation levels correlate with collagen 17 expression in colon tumour samples, and correlate inversely with survival. Finally, this signalling axis enhances the ability of TIC to form tumours in mouse models of malignant lun...
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