Treatment of severe therapy-resistant acute graft-versus-host disease with human adipose tissue-derived mesenchymal stem cells.
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Adipose Tissue↗
Biomedical subjects
B Fang
Publications and source records attributed to B Fang.
Oncolysis and suppression of tumor growth by a GFP-expressing oncolytic adenovirus controlled by an hTERT and CMV hybrid promoter.
One of the challenges of oncolytic virotherapy is the inability to easily track or monitor virus activity during treatment. Here we describe the construction and functional characterization of Ad/hTC-GFP-E1, an oncolytic virus whose transgenes GFP and E1A are both under the control of a synthetic promoter (hTC). This promoter consists of sequences from the human telomorase reverse transcriptase promoter and a minimal cytomegalovirus (CMV) early promoter. The tumor-specific expression of E1A and GFP was demonstrated by Western blot and fluorescent microscope analyses, and the tumor-specific cytotoxicity by crystal-violet staining and cell viability assays. Viral replication and tumor cell lysis occurred at multiplicities of infection (MOI) as low as 100 viral particles per cell in sensitive cell lines. No overt cytotoxic effect was observed in normal human fibroblasts, even at MOIs over 2000 vp. The presence of oncolytic vector was easily visualized and quantitated in vitro and in vivo, in correlation with viral replication. Intralesional administration of the virus into subcutaneous H1299 (NSCLC) tumor xenografts significantly suppressed tumor growth and provided a survival benefit. Together, these results demonstrate that an hTERT-specific oncolytic adenovirus expressing an hTERT-specific transgene is applicable for cancer therapy.
Adenoviridae↗
Combination effect of oncolytic adenovirotherapy and TRAIL gene therapy in syngeneic murine breast cancer models.
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene therapy and oncolytic adenovirotherapy have been investigated extensively in xenografic human tumor models established in immunocompromised nude mice. However, the effects of these therapies on syngeneic murine tumors in immunocompetent settings were not well documented. We hypothesized that TRAIL gene therapy used with an oncolytic adenovirus would overcome the weaknesses of the two therapies used individually. In this study, we evaluated the antitumor effects of an oncolytic adenovirus, Delta24, in both human and murine breast cancer cell lines. We also analyzed the effects of TRAIL gene therapy combined with oncolytic virotherapy in these cancer cells. Our results showed that Delta24 can replicate and help the E1-deleted adenovector replicate in murine cancer cells. We also found that these two therapies combined had greater antitumor activity than either one alone in both human and murine breast cancer cells lines and in the syngeneic breast cancer models established in immunocompetent mice. Moreover, Delta24 virotherapy alone and combined with TRAIL gene therapy dramatically reduced the spontaneous liver metastasis that originated in the subcutaneous 4T1 tumor established in Balb/c mice. These findings provide important considerations in the development and preclinical assessments of oncolytic virotherapy.
Adenoviridae↗
Anticancer activity of 5-benzylidene-2-phenylimino-1, 3-thiazolidin-4-one (BPT) analogs.
We recently identified two compounds of 5-benzylidene-2-phenylimino-1,3-thiazolidin-4-one (BPT) analog, 5-(4-methylbenzylidene)-2-phenylamino-1,3-thiazolidin-4-one (MMPT) and 5-(2,4-dihydroxybenzylidene)-2-phenylimino-1,3-thiazolidin-4-one (DBPT), that can effectively induce apoptosis in cancer cells but not in normal cells, independently of P-glycoprotein status. To further investigate the antitumor activity of BPT analogs, we obtained 18 commercially available analogs of BPT and synthesized 7 analogs in our lab, and analyzed their antitumor activity in various cancer cells, including paclitaxel- and vinorelbine-sensitive and -resistant human lung cancer cells. Two of the compounds were more potent than MMPT or DBPT in induction of apoptosis in certain cancer cell lines and remained tumor selective. Seven compounds did not induce any cytotoxic effects in any of the cell lines tested at the highest concentration tested (31 microM). The other compounds induced cytotoxic effects in some cancer cells but not in others or were less potent than MMPT and DBPT. Cell uptake studies showed that analogs that effectively induced cell killing in paclitaxel- and vinorelbine-resistant cells could be taken up easily by those cells despite their high levels of P-glycoprotein expression. These data further demonstrate that thiazolidinone analogs are not P-glycoprotein substrates and could be useful for treatment of P-glycoprotein overexpressing refractory cancers.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Oncolytic virotherapy for cancer treatment: challenges and solutions.
Advances in gene modification and viral therapy have led to the development of a variety of vectors in several viral families that are capable of replication specifically in tumor cells. Because of the nature of viral delivery, infection, and replication, this technology, oncolytic virotherapy, may prove valuable for treating cancer patients, especially those with inoperable tumors. Current limitations exist, however, for oncolytic virotherapy. They include the body's B and T cell responses, innate inflammatory reactions, host range, safety risks involved in using modified viruses as treatments, and the requirement that most currently available oncolytic viruses require local administration. Another important constraint is that genetically enhanced vectors may or may not adhere to their replication restrictions in long-term applications. Several solutions and strategies already exist, however, to minimize or circumvent many of these limitations, supporting viral oncolytic therapy as a viable option and powerful tool in the fight against cancer.
Animals↗
JNK1-dependent antimitotic activity of thiazolidin compounds in human non-small-cell lung and colon cancer cells.
We recently identified two thiazolidin compounds, 5-[(4-methylphenyl)methylene]-2-(phenylamino)-4(5H)-thiazolone (MMPT) and 5-(2,4-dihydroxybenzylidene)-2-(phenylimino)-1,3-thiazolidin (DBPT), that inhibit the growth of human non-small-cell lung and colon cancer cells independent of P-glycoprotein and p53 status. Here we further investigated the mechanism by which these thiazolidin compounds mediate their anticancer effects. Treatment of cancer cells with MMPT and DBPT led to a time-dependent accumulation of cells arrested in the G2/M phase with modulation of the expression of proteins such as cyclin B1, cdc25C, and phosphorylated histone H3. Moreover, treatment with MMPT and DBPT increased M-phase arrest with abnormal spindle formation. DBPT-mediated G2/M phase arrest and phosphorylation of cdc25C and histone H3 were abrogated when JNK activation was blocked either with SP600125, a specific JNK inhibitor, or a dominant-negative JNK1 gene. Moreover, DBPT-mediated microtubule disruption was also blocked by SP600125 treatment. Our results demonstrate that thiazolidin compounds can effectively induce G2/M arrest in cancer cells and that this G2/M arrest requires JNK activation.
Aniline Compounds↗
Differential involvement of Bax and Bak in TRAIL-mediated apoptosis of leukemic T cells.
TRAIL-induced apoptosis has been considered a promising therapeutic approach for tumors that are resistant to chemotherapy, which is usually mediated via mitochondrial apoptotic cascades. Recent studies have shown that in certain cancer cells, TRAIL-mediated apoptosis is also dependent on mitochondrial involvement, suggesting that similar mechanisms of resistance to chemotherapy might be implicated in the resistance of tumor cells to TRAIL. We have used TRAIL-resistant leukemic cells that are deficient in both Bax and Bak to determine the roles of these Bcl-2 members in TRAIL-mediated apoptosis. Exposure of these cells to TRAIL did not have an impact on cell viability, although it induced the processing of caspase-3 to its active p20 subunit. The activity of the p20 caspase-3 appeared to be inhibited as no autoprocessing of this p20 subunit or cleavage of known caspase-3 substrates were detected. Also, in the absence of Bax and Bak, no release of mitochondrial apoptogenic proteins was observed following TRAIL treatment. Adenoviral transduction of the Bax, but not the Bak gene, to the Bax/Bak-deficient leukemic cells rendered them TRAIL-sensitive as assessed by enhanced apoptotic death and caspase-3 processing. These findings demonstrate preferential utilization of Bax over Bak in leukemic cell response to specific apoptotic stimulation.
Adenoviridae↗
[Adenoviral vector expressing the TRAIL gene driven by the hTERT promoter].
BACKGROUND: Resistance can be overcome by modified adenoviral vectors containing an Arg-Gly-Asp (RGD) sequence. We constructed an adenoviral vector with RGD-modified fibers, expressing the TRAIL gene from the human telomerase reverse transcriptase (hTERT) promoter (designated Ad/TRAIL-F/RGD), and evaluated its antitumor activity in vitro and in vivo. METHODS: The induction of apoptosis by the new vector Ad/TRAIL-F/RGD was evaluated in human carcinoma cells derived from hepatocellular carcinoma (Hep G2, Hep 3b), pancreatic carcinoma (Panc-1, Capan-1), and colon carcinoma (LOVO, SW 620) . Cell viability was measured by the XTT assay and GFP expression and apoptosis induction by fluorescence-activated cell sorting (FACS) and Western blot. In vivo experiments were performed in an orthotopic pancreas tumor model in nu/nu nude mice. RESULTS: Treatment with Ad/TRAIL-F/RGD and Ad/gTRAIL resulted in significantly reduced cell viability in comparison to PBS and Ad/CMV-GFP treatment in all examined human carcinoma cell lines. In addition, mice treated with Ad/TRAIL-F/RGD showed a significantly decreased tumor growth than both control groups. CONCLUSIONS: Our results suggest that Ad/TRAIL-F/RGD may become a potent therapeutic agent for the treatment of different human solid carcinomas.
Adenoviruses, Human↗
Molecular therapy for peritoneal dissemination of xenotransplanted human MKN-45 gastric cancer cells with adenovirus mediated Bax gene transfer.
BACKGROUND: Gene therapy is an innovative therapeutic approach for cancer. An adenoviral vector expressing the tumour suppressor p53 gene (Ad/p53) is currently under clinical evaluation for various cancers. We recently developed a binary adenoviral vector system that can express the strong proapoptotic gene Bax (Ad/PGK-GV16+Ad/GT-Bax: Ad/Bax). AIMS: To evaluate the potential of Bax gene therapy for gastric cancer, we assessed its antitumour effect in comparison with that of p53. METHODS: The human gastric cancer cell lines MKN-1, MKN-7, MKN-28, and MKN-45 were treated with Ad/Bax or Ad/p53, and cell viability, transgene expression, and caspase activation were assessed in vitro. To compare the antitumour effects of Ad/Bax and Ad/p53 treatment in vivo, subcutaneous tumours and peritoneal dissemination of MKN-45 cells were generated in nude mice. Each mouse underwent intratumoral or intraperitoneal administration of viruses and the growth of implanted tumours was observed after treatment. RESULTS: Treatment with Ad/Bax and Ad/p53 resulted in marked Bax and p53 protein expression and effective apoptosis induction in MKN-1, MKN-7, and MKN-28 cells in vitro. In contrast, MKN-45 cells showed resistance to Ad/p53 and only treatment with Ad/Bax resulted in activation of caspase 3 expression and massive apoptosis. Ad/Bax treatment was more effective in suppressing both subcutaneous and peritoneally disseminated MKN-45 tumours compared with Ad/p53 treatment. CONCLUSION: Ad/Bax treatment significantly inhibited the growth of even p53 resistant gastric cancer in vitro and in vivo. Therefore, adenovirus mediated Bax gene transfer may be useful in gene therapy for gastric cancers.
Adenoviridae↗
TRAIL-beta and TRAIL-gamma: two novel splice variants of the human TNF-related apoptosis-inducing ligand (TRAIL) without apoptotic potential.
Tumour necrosis factor (TNF) related apoptosis-inducing ligand (TRAIL/APO2L) is a recently identified member of the TNF family, which induces programmed cell death in a variety of neoplastic cell types, but not in most nonneoplastic cells. In this study, we report on the identification of two novel alternative splice variants of TRAIL in neoplastic and non-neoplastic human cells lacking either exon 3 (TRAIL-beta) or exons 2 and 3 (TRAIL-gamma). In both splice variants, loss of exon 3 resulted in a frame shift generating a stop codon with consecutive extensive truncation in the extracellular domain. Ectopic expression revealed a loss of proapoptotic potential for both alternative splice variants. In contrast to the predominantly cytoplasmatic localisation of GFP-tagged TRAIL-alpha and TRAIL-beta, TRAIL-gamma showed an additional association with the cell surface and nuclear membrane. In conclusion, alternative splicing might be involved in fine tuning of TRAIL-induced apoptosis and underlines the complexity of the TRAIL system.
Alternative Splicing↗
hTERT promoter induces tumor-specific Bax gene expression and cell killing in syngenic mouse tumor model and prevents systemic toxicity.
We recently showed that the human telomerase reverse transcriptase (hTERT) promoter induces tumor-specific Bax gene expression and selectively kills various human cancer cells both in vitro and in xenograft tumors. However, it remains unclear whether the hTERT promoter can be used to induce transgene expression in syngenic tumors in mice and whether Bax gene expression driven by the hTERT promoter will cause long-term, stem cell-related toxicity. To address these questions, we tested hTERT promoter-driven, adenovirus-mediated Bax transgene expression in an established syngenic mouse tumor model and its effects on tumor and normal murine tissues. The hTERT promoter was highly active in several murine tumor cell lines and a transformed cell line, but not in non-transformed and normal murine cell lines. The hTERT promoter induced tumor-specific Bax gene expression in mouse UV-2237m fibrosarcoma cells both in vitro and in vivo and suppressed syngenic tumor growth in immune-competent mice with no obvious acute or long-term toxic effects. Moreover, hTERT promoter-driven transgene expression in human CD34(+) bone marrow progenitor cells had effects similar to those observed in other normal human cells, suggesting that the hTERT promoter is much less active in CD34(+) cells than in tumor cells. Together, our data demonstrate that the hTERT promoter may allow the use of proapoptotic genes for cancer treatment without noticeable effects on progenitor cells.
Adenoviridae↗
An effective immunization and cancer treatment with activated dendritic cells transduced with full-length wild-type p53.
P53-based immunization is an attractive approach to cancer immunotherapy due to the accumulation of p53 protein in tumor, but not in normal cells. However, it was not known whether immune response against self-protein (p53) could be generated in vivo. Mouse dendritic cells (DCs) were transduced with adenoviral construct containing murine full-length wild-type p53 (Ad-p53). Repeated immunizations with these cells protected 60% of mice against challenge with MethA sarcoma cells bearing point mutations in p53 gene. Activation of DCs via ligation of CD40 significantly improved the results of immunization: all mice were protected against MethA sarcoma. The treatment of MethA tumor-bearing mice with activated Ad-p53-transduced DCs showed complete tumor rejection in four out of six mice. The specificity of antitumor immune response was confirmed by CTL assay. The analysis of phenotype and function of DCs demonstrated that the effect of CD40 ligation on these cells was enhanced by their infection with Ad-p53. The level of neutralizing anti-adenovirus antibody was moderately elevated in these mice. No signs of autoimmune reaction were evident during detailed pathological evaluation of treated mice. These data demonstrate that activated Ad-p53-infected DCs are able to break tolerance to this protein and can be used in immunotherapy of cancer.
Adenoviridae↗
Mechanisms involved in development of resistance to adenovirus-mediated proapoptotic gene therapy in DLD1 human colon cancer cell line.
To evaluate resistance that develops in cancer cells during treatment with adenoviral vectors expressing proapoptotic genes, we repeatedly treated the human colon cancer cell line DLD1 with adenoviral vectors expressing the human Bax gene and the human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene. DLD1 cells resistant to the Bax- or TRAIL-expressing adenoviral vectors were then selected and designated as DLD1/Bax-R or DLD1/TRAIL-R cells, respectively. Further study showed that resistance in DLD1/Bax-R cells was caused by resistance to adenoviral infection, which can be overcome by dose escalation of the adenoviral vectors. However, resistance in DLD1/TRAIL-R cells was caused by resistance to the TRAIL gene. Therefore, different mechanisms are involved in the development of resistance during adenovirus-mediated proapoptotic gene therapy. A survey of molecules involved in TRAIL- or Bax-mediated apoptotic pathways showed no significant change in expression of death receptors, death decoy receptors; FLIP; Bcl-2; Bcl-xS; Bax; Bak; XIAP or caspase-2, -7, -8, or -9 in either DLD1/Bax-R or DLD1/TRAIL-R cells. Bcl-xL expression detected in both mRNA and protein level assays was three times higher in DLD1/TRAIL-R cells than in parental or DLD1/Bax-R cells. However, transfection of DLD1 cells with the Bcl-xL gene showed that overexpression of Bcl-xL is not sufficient for the resistance. Moreover, DLD1/Bax-R cells were sensitive to adenoviral vectors that expressed the TRAIL gene, but resistant to adenoviral vectors that expressed the Bak gene. In contrast, DLD1/TRAIL-R cells were sensitive to adenoviral vectors that expressed either Bax or Bak gene. Thus, alternative application of adenoviral vectors that expressed proapoptotic genes in different pathways or different cell killing models may delay or prevent development of resistance in adenovirus-mediated proapoptotic gene therapy.
Adenoviridae↗
Combined TRAIL and Bax gene therapy prolonged survival in mice with ovarian cancer xenograft.
We evaluated the antitumor activity of the Bax gene and green fluorescent protein/tumor necrosis factor-related apoptosis-inducing ligand (GFP/TRAIL) fusion gene driven by the human telomerase reverse transcriptase promoter both separately and combined in the human ovarian cancer lines SKOV3ip and DOV13 and human lung cancer line H1299. In vitro study showed that both TRAIL- and Bax-expressing vectors elicited significant cell killing in H1299 and SKOV3ip cells, but only the GFP/TRAIL gene elicited significant cell killing in DOV13 cells. Combined TRAIL and Bax therapy also produced more profound cell killing in SKOV3ip and H1299 cells, but not DOV13 cells without escalation of the vector doses. To further evaluate the combined effects of Bax and TRAIL, abdominally spread tumors were established in nude mice via intraperitoneal inoculation of SKOV3ip cells followed by that of adenoviral vectors. Tumor growth, ascites formation, survival duration and toxicity were evaluated after treatment. We found that treatment using the Bax- or TRAIL-expressing vector alone significantly suppressed tumor growth and ascites formation, and prolonged animal survival when compared with that of using PBS or a control vector. Combined TRAIL and Bax therapy further prolonged survival significantly when compared with therapy using the TRAIL or Bax gene alone. Transgene expression and apoptosis induction were not detected in normal human ovarian epithelial cells in vitro or normal mouse tissues in vivo after intraperitoneal vector administration. Also, liver toxicity was not detected after either treatment. Thus, combined TRAIL and Bax gene therapy may be useful for treatment of abdominally spread tumors.
Animals↗
Targeting HER-2/neu-overexpressing breast cancer cells by an antisense iron responsive element-directed gene expression.
Overexpression of HER-2/neu proto-oncogene is found in many human cancers including 20-30% of breast cancer and is a predictor of poor prognosis. To target breast cancer cells that overexpress HER-2/neu mRNA, we previously described a novel strategy that combines the principle of antisense (AS) and translational inhibitory activity conferred by an iron-responsive element (IRE) (AS-IRE). Here, we showed that three potential AS-IREs, i.e. AS-IRE1, 4, and 5, derived from HER-2/neu antisense sequence could bind endogenous iron regulatory protein (IRP) and, when placed in 5' untranslated region (5'UTR) of a reporter gene, the gene expression could be translationally repressed by recombinant IRP in vitro. Using AS-IRE4 as our model, we demonstrated that it is regulated by iron, and importantly, such regulation is impaired in HER-2/neu-overexpressing breast cancer cells. Furthermore, we showed that AS-IRE4 could preferentially direct the expression of a reporter gene in HER-2/neu-overexpressing breast cancer cells. Interestingly, when AS-IRE4 was placed in 5'UTR of Bax gene, a pro-apoptotic protein in the Bcl-2 protein family, we observed a preferential cell killing in breast cancer cells that overexpress HER-2/neu. Taken together, our results suggest that AS-IRE behaves as a functional IRE and it may direct therapeutic gene expression to preferentially target HER-2/neu-overexpressing breast cancer cells.
Antisense Elements (Genetics)↗
Resistance to granzyme B-mediated cytochrome c release in Bak-deficient cells.
Granzyme B (GrB), a serine protease with substrate specificity similar to the caspase family, is a major component of granule-mediated cytotoxicity of T lymphocytes. Although GrB can directly activate caspases, it induces apoptosis predominantly via Bid cleavage, mitochondrial outer membrane permeabilization, and cytochrome c release. To study the molecular regulators for GrB-mediated mitochondrial apoptotic events, we used a CTL-free cytotoxicity system, wherein target cells are treated with purified GrB and replication-deficient adenovirus (Ad). We report here that the Bcl-2 proapoptotic family member, Bak, plays a dominant role in GrB-mediated mitochondrial apoptotic events. A variant of Jurkat cells, deficient in Bak expression, was resistant to GrB/Ad-mediated apoptosis, as determined by lack of membranous phosphatidylserine exposure, lack of DNA breaks, lack of mitochondrial outer membrane permeabilization, and unchanged expression of inner mitochondrial membrane cardiolipin. The resistance of Bak-deficient cells to GrB/Ad cytotoxicity was reversed by transduction of the Bak gene into these cells. The requirement for both Bid and Bak, was further demonstrated in a cell-free system using purified mitochondria and S-100 cytosol. Purified mitochondria from Bid knockout mice, but not from Bax knockout mice, failed to release cytochrome c in response to autologous S-100 and GrB. Also, Bak-deficient mitochondria did not release cytochrome c in response to GrB-treated cytosol unless recombinant Bak protein was added. These results are the first to report a role for Bak in GrB-mediated mitochondrial apoptosis. This study demonstrates that GrB-cleaved Bid, which differs in size and site of cleavage from caspase-8-cleaved Bid, utilizes Bak for cytochrome c release, and therefore, suggests that deficiency in Bak may serve as a mechanism of immune evasion for tumor or viral infected cells.
Adenoviridae↗
Use of the probasin promoter ARR2PB to express Bax in androgen receptor-positive prostate cancer cells.
BACKGROUND: Adenovirus-mediated overexpression of the apoptosis-inducing protein Bax can induce apoptosis in prostate cancer cell lines. Constitutive overexpression of Bax could result in unwanted apoptosis in every site of accidental Bax accumulation in vivo. Therefore, we developed an adenoviral construct (Av-ARR2PB-Bax) in which the probasin promoter, modified to contain two androgen response elements, drives Bax expression. This promoter would be expected to limit expression of Bax to cells expressing the androgen receptor. METHODS: A variety of androgen receptor (AR)-positive and -negative cell lines of prostatic or nonprostatic origin were infected with Av-ARR2PB-Bax or a control virus, Av-ARR2PB-CAT, in which the same promoter drives expression of the chloramphenicol acetyl transferase-reporter gene. Bax expression and apoptosis in vitro were assessed by western blot analysis. Tumor size and apoptosis in vivo were assessed after four weekly injections of Av-ARR2PB-Bax or Av-ARR2PB-CAT into subcutaneous LNCaP xenografts growing in uncastrated male mice. All statistical tests were two-sided. RESULTS: Bax was overexpressed in an androgen-dependent way in AR-positive cell lines of prostatic origin but not in AR-positive cells of nonprostatic origin or in AR-negative cell lines of either prostatic or nonprostatic origin. The androgen dihydrotestosterone activated apoptosis in LNCaP cells infected with Av-ARR2PB-Bax but not in those infected with Av-ARR2PB-CAT. Av-ARR2PB-Bax-injected LNCaP xenograft tumors decreased in tumor size from 34.1 mm3 (95% confidence interval [CI] = 25.1 mm3 to 43.1 mm3) to 24.6 mm3 (95% CI = -2.5 mm3 to 51.7 mm3), but the difference was not statistically significant (P =.5). Tumors injected with Av-ARR2PB-CAT increased in size, from 28.9 mm3 (95% CI = 12.7 mm3 to 45.1 mm3) to 206 mm3 (95% CI = 122 mm3 to 290 mm3) (P =.002) and contained statistically significant more apoptotic cells (23.3% [95% CI = 21.1% to 25.6%] versus 9.5% [95% CI = 8.0% to 11.1]) (P<.001). CONCLUSIONS: Av-ARR2PB-Bax induces androgen-dependent therapeutic apoptosis in vitro and in vivo by activating apoptosis in AR-positive cells derived specifically from prostatic epithelium and does not affect nonprostatic cells.
Adenoviridae↗
On the trail of a killer. Slow to respond, China faces a spreading HIV-AIDS epidemic.
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Acquired Immunodeficiency Syndrome↗