Caspase-10: a molecular switch from cell-autonomous apoptosis to communal cell death in response to chemotherapeutic drug treatment.

The mechanisms of how chemotherapeutic drugs lead to cell cycle checkpoint regulation and DNA damage repair are well understood, but how such signals are transmitted to the cellular apoptosis machinery is less clear. We identified a novel apoptosis-inducing complex, we termed FADDosome, which is driven by ATR-dependent caspase-10 upregulation. During FADDosome-induced apoptosis, cFLIPL is ubiquitinated by TRAF2, leading to its degradation and subsequent FADD-dependent caspase-8 activation. Cancer cells lacking caspase-10, TRAF2 or ATR switch from this cell-autonomous suicide to a more effective, autocrine/paracrine mode of apoptosis initiated by a different complex, the FLIPosome. It leads to processing of cFLIPL to cFLIPp43, TNF-α production and consequently, contrary to the FADDosome, p53-independent apoptosis. Thus, targeting the molecular levers that switch between these mechanisms can increase efficacy of treatment and overcome resistance in cancer cells.

DR4 specific TRAIL variants are more efficacious than wild-type TRAIL in pancreatic cancer.

Current treatment modalities for pancreatic carcinoma afford only modest survival benefits. TRAIL, as a potent and specific inducer of apoptosis in cancer cells, would be a promising new treatment option. However, since not all pancreatic cancer cells respond to TRAIL, further improvements and optimizations are still needed. One strategy to improve the effectiveness of TRAIL-based therapies is to specifically target one of the 2 cell death inducing TRAIL-receptors, TRAIL-R1 or TRAIL-R2 to overcome resistance. To this end, we designed constructs expressing soluble TRAIL (sTRAIL) variants that were rendered specific for either TRAIL-R1 or TRAIL-R2 by amino acid changes in the TRAIL ectodomain. When we expressed these constructs, including wild-type sTRAIL (sTRAIL(wt)), TRAIL-R1 (sTRAIL(DR4)) and TRAIL-R2 (sTRAIL(DR5)) specific variants, in 293 producer cells we found all to be readily expressed and secreted into the supernatant. These supernatants were subsequently transferred onto target cancer cells and apoptosis measured. We found that the TRAIL-R1 specific variant had higher apoptosis-inducing activity in human pancreatic carcinoma Colo357 cells as well as PancTu1 cells that were additionally sensitized by targeting of XIAP. Finally, we tested TRAIL-R1 specific recombinant TRAIL protein (rTRAIL(DR4)) on Colo357 xenografts in nude mice and found them to be more efficacious than rTRAIL(wt). Our results demonstrate the benefits of synthetic biological approaches and show that TRAIL-R1 specific variants can potentially enhance the therapeutic efficacy of TRAIL-based therapies in pancreatic cancer, suggesting that they can possibly become part of individualized and tumor specific combination treatments in the future.

Both human and mouse mesenchymal stem cells promote breast cancer metastasis.

Cell therapy has the potential to offer novel treatment modalities for a number of diseases including cancer, and stem cells and in particular mesenchymal stem cells (MSCs) have been experimentally used to deliver therapeutic transgenes. However, conflicting reports have on the one side found that human MSCs can promote metastasis, while on the other hand other studies have shown that MSCs can stall the growth of metastatic lesions. In order to clarify the role of MSCs in metastasis development, we tested whether murine MSCs would behave similarly to human cells in mice. We found that the tissue distribution of human and mouse MSCs was nearly identical after intravenous injection. In mice with MDA-MB-231 mammary carcinoma xenografts we found that a fraction of MSCs infiltrated the primary tumor mass, but that the general tissue distribution of MSCs was unaffected by the tumor-burden. About half of the tumor-burdened animals that were treated with murine and human MSCs, respectively, harbored metastatic lesions with only 17% of controls showing metastatic nodules. Hence, both human and mouse MSCs possess metastasis-promoting activity raising concerns about the safe use of MSCs, but at the same time making the use of murine transgenic model systems feasible to study the role of MSCs in metastasis development and possibly finding ways of using them safely as cell therapeutic vehicles.

Targeting of XIAP combined with systemic mesenchymal stem cell-mediated delivery of sTRAIL ligand inhibits metastatic growth of pancreatic carcinoma cells.

Disseminating tumors are one of the gravest medical problems. Here, we combine the tumor-specific apoptosis-inducing activity of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with the ability of mesenchymal stem cells (MSCs) to infiltrate both tumor and lymphatic tissues to target primary tumors as well as disseminated cancer cells in a human pancreatic cancer mouse model. Furthermore, we targeted X-linked inhibitor of apoptosis protein (XIAP) by RNA interference (RNAi) inside the cancer cells to make use of the apoptosis sensitization as well the antimetastatic effect that is afforded by XIAP silencing. We generated MSCs, termed MSC.sTRAIL, that express and secrete a trimeric form of soluble TRAIL (sTRAIL). MSC.sTRAIL triggered limited apoptosis in human pancreatic carcinoma cells that were resistant to soluble recombinant TRAIL, which is most likely due to the enhanced effect of the direct, cell-mediated delivery of trimeric TRAIL. MSC.sTRAIL-mediated cell death was markedly increased by concomitant knockdown of XIAP by RNAi in the cancer cells. These findings were confirmed in xenograft models, in which tumors from the parental pancreatic carcinoma cells showed only growth retardation on treatment with MSC.sTRAIL, whereas tumors with silenced XIAP that were treated with MSC.sTRAIL went into remission. Moreover, animals with XIAP-negative xenografts treated with MSC.sTRAIL were almost free of lung metastasis, whereas animals treated with control MSCs showed substantial metastatic growth in the lungs. In summary, this is the first demonstration that a combined approach using systemic MSC-mediated delivery of sTRAIL together with XIAP inhibition suppresses metastatic growth of pancreatic carcinoma.

T-2 toxin-induced apoptosis in rat keratinocyte primary cultures.

T-2 toxin, a kind of trichothecene mycotoxins produced by the genus Fusarium, induces apoptosis in basal keratinocytes when topically applied to the dorsal skin of rats. In the present study, direct effects of T-2 toxin on keratinocyte primary cultures obtained from newborn rats were examined after the third passage. Keratinocyte medium containing 0.25 microg/ml of T-2 toxin dissolved in dimethyl sulfoxide or solvent alone was added to 4-day cultures and incubated at 37 degrees C. At 0.5, 1, 3, 5, 7, and 9 h after treatment (h), feeder layer was separated from flasks, and cells were trypsinized. Cell viability was estimated by trypan blue exclusion method. In addition, RNA was obtained and RT-PCR was performed. Samples obtained from slide cultures at 3, 6, 9, and 12 h were fixed in 4% paraformaldehyde or 2.5% glutaraldehyde for morphological examination. After T-2 toxin application, cell viability decreased to 40% at 3 h. At 6 h, small-sized keratinocytes showed pyknosis or karyorrhexis, resulting in detachment from slides. The number of such cells increased until 12 h. These small-sized keratinocytes showed ultrastructural changes characteristic for apoptosis. At the same time, large squamous keratinocytes showed intracytoplasmic edema. The expression of apoptosis-related genes (c-fos and c-jun) and cytokines (TNF-alpha and IL-1beta) mRNAs markedly increased before the development of apoptosis. These findings indicate that c-fos and c-jun oncogenes and TNF-alpha and IL-1beta play an important role in the development of T-2 toxin-induced apoptosis in keratinocytes.

Detection of porcine circovirus 2 (PCV-2) DNA by nested PCR from formalin-fixed tissues of post-weaning multisystemic wasting syndrome (PMWS) pigs in Thailand.

A retrospective study for the detection of porcine circovirus 2 (PCV-2) DNA was conducted by nested PCR method in 16 cases of swine post-weaning multisystemic wasting syndrome (PMWS) in Thailand. Histopathology showed characteristic lesions of PMWS and intracytoplasmic viral inclusion bodies in macrophages infiltrating in lymphoid tissues. PCV-2 DNA was detected from formalin-fixed and/or formalin-fixed paraffin-embedded tissues from all pigs with PMWS. The amplified products were digested with Hae III.

Mitotic activity of the duodenal crypt enterocytes in mice transplanted with EA21a mammary carcinoma.

The presence of a tumor generally changes the mitotic activity of the normal cell population in mice. In the present work, the mitotic activity of the duodenal crypt enterocytes in EA21a mammary carcinoma-bearing mice was determined. The results show that there is a patent circadian variation in normal mice and, in the presence of the EA21a mammary tumor, cell proliferation is stimulated. Stimulation was evident in enterocytes from the intermediate as well as the superficial regions of the crypt. Some humoral factors produced by the transplanted tumor could interfere with the regulatory mechanism of the mitotic activity of duodenal crypt enterocytes.