Mechanisms involved in development of resistance to adenovirus-mediated proapoptotic gene therapy in DLD1 human colon cancer cell line

Cancer Biology & Therapy

Davis

et al. 2004

Activation of MAP kinases is involved in various cellular processes, including immunoregulation, inflammation, cell growth, cell differentiation, and cell death. To investigate the role of p38 MAP kinase activation in the signaling pathway of TRAIL-mediated apoptosis, we compared TRAIL-mediated MAP kinase activation in TRAIL-susceptible human colon cancer cell line DLD1 and TRAIL-resistant DLD1/TRAIL-R cells. TRAIL-mediated activation of ERK occurred in both cell lines. In contrast, both DLD1 and DLD1/TRAIL-R cells showed no obvious JNK activation after treatment with TRAIL. Interestingly, TRAIL-mediated activation of p38 MAP kinases was observed in DLD1 cells but not in DLD1/TRAIL-R cells. However, activation of p38 MAP kinases was observed in both DLD1 and DLD1/TRAIL-R cells after treatment with anisomycin. Furthermore, inhibiting activated p38 MAP kinases with known inhibitors or with an adenovector expressing dominant negative p38α did not block TRAIL-mediated cell death in DLD1 cells. Moreover, activation of p38 MAP kinases by adenovectors expressing constitutive MKK3 or MKK6 (Ad/MKK3bE or Ad/MKK6bE) did not induce cell death in either DLD1 or DLD1/TRAIL-R cell lines. Our results suggest that activation of p38 MAP kinases does not play a major role in TRAIL-mediated apoptosis in DLD1 cells and that lack of TRAIL-mediated p38 MAP kinase activation may not be the mechanism of TRAIL-resistance in DLD1/TRAIL-R cells. INTRODUCTIONThe tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) superfamily. Like the other two members of this superfamily TNF-α and Fas ligand, TRAIL has strong antitumor activity both in vitro and in vivo in a wide range of cancer cell types. 1 Unlike TNF-α and Fas ligand, however, TRAIL exhibits minimal cytotoxity to most of normal cells and tissues and, therefore, shows potential of its becoming a new therapeutic agent for cancer treatment. [2][3][4] For most cell types, TRAIL induces cell death through apoptosis. Although the detailed mechanisms of the signaling pathway of TRAIL-induced apoptosis are still unclear, some important components and steps in the signaling pathway have already been elucidated. The interaction of TRAIL and its two death receptors, DR4 (TRAIL-R1) and/or DR5 (TRAIL-R2), is the initial step of TRAIL-induced apoptosis. 5,6 The binding of TRAIL to its receptors leads to trimerization and activation of the death receptors. The activated death receptors recruit and activate an adaptor protein called FADD (Fas-associated death domain), which in turn recruits and activates caspase-8, leading to the formation of the death-inducing signaling complex (DISC). 7,8 Once caspase-8 is activated, it can lead to apoptosis either by activating effector caspase-3 and -7, which in turn act on final death substrates, 8 or by activating Bid, which induces the accumulation of Bax in mitochondria, the release of cytochrome c from mitochondria, the activation of caspases-9, -3, and -7, and finally programmed cell death. 9,...

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

Lack of p38 MAP Kinase Activation in TRAIL-Resistant Cells is Not Related to the Resistance to TRAIL-Mediated Cell Death

Zhang

Cancer Biology & Therapy

Davis

et al. 2004

“…The DLD1/Bcl-XL cells were derived from DLD-1 cells by stable transfection with the Bcl-XL gene as described previously (Zhang et al, 2002) and AT22IJEpEBS7 was originally constructed in the laboratory of Dr Yosef Shiloh (Ziv et al, 1997). All cells were maintained in RPMI-1640 or Dulbecco's modified Eagle's medium plus 5% (v/v) heatinactivated fetal bovine serum, 1% glutamine, and 1% antibiotics.…”

Section: Cells and Cell Culturementioning

confidence: 99%

“…The cells were then fixed with cold 70% ethanol overnight at 41C. At 30 min before the assay, propidium iodide staining was performed as described previously (Kagawa et al, 2001;Zhang et al, 2002). For the apoptosis assay, the floating cells were included.…”

Section: Flow Cytometry Assaymentioning

confidence: 99%

“…A measure of 1 mg of total RNA from each sample was reverse-transcribed into cDNA by using random hexamers as reverse transcription primers (TaqMan Reverse Transcription Reagents, Applied Biosystems). Then a typical real-time PCR was performed as described previously (Zhang et al, 2002). The human GAPDH gene was used as an internal control for normalization of the mRNA amount.…”

Section: Western Blot Analysismentioning

confidence: 99%

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Induction of S-phase arrest and p21 overexpression by a small molecule 2[[3-(2,3-dichlorophenoxy)propyl] amino]ethanol in correlation with activation of ERK

Zhang

et al. 2004

Oncogene

We recently found that a small molecule 2 [[3-(2,3-dichlorophenoxy)propyl]amino]ethanol (2,3-DCPE) could induce apoptosis and downregulate Bcl-XL expression in various cancer cells. Here, we found that 2,3-DCPE suppressed the proliferation of Bcl-XL-overexpressing cancer cells without inducing apoptosis. Subsequently, we found that 2,3-DCPE could induce S-phase arrest and upregulate p21 but not p27 at a time-and dose-dependent but p53-dispensable manner in DLD-1 human colon cancer cells. Activation of ERK was also detected after treatment with 2,3-DCPE. Moreover, p21 induction was dramatically attenuated by ERK inhibitors PD98059 and U0126. Induction of p21 and S-phase arrest and corresponding activation of ERK were also observed in ATM-defective cells, suggesting that 2,3-DCPE-induced these events were ATM-dispensable. Furthermore, ERK inhibitors dramatically attenuated 2,3-DCPE-induced Sphase arrest. Together, our data indicate that ERK activation correlated with the 2,3-DCPE-mediated induction of p21 expression and S-phase arrest. This finding may have implication for cancer therapy. Oncogene (2004Oncogene ( ) 23, 4984-4992. doi:10.1038 Published online 3 May 2004Keywords: S-phase arrest; p21; ATM; p53; ERK; small molecule IntroductionCell cycle progression is tightly controlled by various cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors, and certain tumor suppressor gene products, such as p53 and RB protein (Sherr, 1996). Deregulation of the cell cycle is one of the critical events that drive cancer cells into uncontrolled proliferation (Evan and Vousden, 2001). Molecular changes, including the overexpression of cyclins and CDKs and the loss of CDK inhibitors and tumor-suppressor proteins resulting from gene mutations or epigenetic inactivation, are frequently detected in tumor cells (Sherr, 1996;Malumbres and Barbacid, 2001). Because of the important roles of cell cycle deregulation in tumorigenesis and tumor progression, molecules involved in cell cycle regulation also serve as potential targets for therapeutic intervention in cancers.CDKs, the central players in cell cycle progression, are the ultimate targets of many anticancer investigations. Strategies explored to inhibit CDKs for cancer therapy include use of the small molecule CDK inhibitors (Senderowicz, 2000); gene therapy to restore the functions of CDK inhibitors, such as p21 (Eastham et al., 1995;Hall et al., 2000), p16 (Schreiber et al., 1999), and p27 (Katayose et al., 1997); modulating the expression of CDK inhibitors by small molecules (Lodygin et al., 2002); and suppressing the expression of cyclin D1 or cyclin E1 by antisense or small interfering RNA (siRNA) technologies (Kornmann et al., 1998). Ectopic expression of CDK inhibitors by gene therapy was found to inhibit tumor growth in preclinical studies (Eastham et al., 1995;Katayose et al., 1997;Schreiber et al., 1999). Meanwhile, certain small molecule modulators of CDKs are under clinical trials (Senderowicz, 2000).We recently found that a synthetic compound, 2[[3-(2,3-dichloro...

Adenovirus‐mediated small hairpin RNA targeting Bcl‐XL as therapy for colon cancer

Hu³

et al. 2007

Intl Journal of Cancer

TXBcl-XL, an anti-apoptotic protein of Bcl-2 family, is overexpressed in colon cancers. To determine Bcl-XL's potential feasibility as a therapeutic target, we constructed a recombinant adenovirus that expressed a U6 promoter-driven small hairpin RNA (shRNA) targeting Bcl-XL (Ad/Bcl-XL shRNA) and evaluated the vector's ability to induce RNA interference in vivo and alter apoptosis induction in colon cancer cells and tumours. Ad/Bcl-XL shRNA effectively knocked down Bcl-XL expression in colon cancer cells and decreased their viability. Treatment with Ad/Bcl-XL shRNA but not control vectors led to dramatically increased cleavage of cellular apoptosis-related enzymes caspase-9, caspase-3 and poly (ADP-ribose) polymerase. Ad/Bcl-XL shRNA also significantly suppressed the growth of subcutaneous tumours derived from DLD1 cells in a nude mouse model and did so without causing any obvious damage to normal tissues or normal human fibroblasts. Together, our results support the feasibility of using adenovirusmediated RNA interference therapy targeting Bcl-XL against colon cancers and warrant further studies of its safety and efficacy. ' 2007 Wiley-Liss, Inc.