In cancer, combinations of drugs targeting different cellular functions is well accepted to improve tumor control. We studied the effects of a Pseudomonas exotoxin A (PE) - based immunotoxin, the 9.2.27PE, and the BH-3 mimetic compound ABT-737 in a panel of melanoma cell lines. The drug combination resulted in synergistic cytotoxicity, and the cell death observed was associated with apoptosis, as activation of caspase-3, inactivation of Poly (ADP-ribose) polymerase (PARP) and increased DNA fragmentation could be prevented by pre-treatment with caspase and cathepsin inhibitors. We further show that ABT-737 caused endoplasmic reticulum (ER) stress with increased GRP78 and phosphorylated eIF2α protein levels. Moreover, treatment with ABT-737 increased the intracellular calcium levels, an effect which was enhanced by 9.2.27PE, which as a single entity drug had minimal effect on calcium release from the ER. In addition, silencing of Mcl-1 by short hairpin RNA (shRNA) enhanced the intracellular calcium levels and cytotoxicity caused by ABT-737. Notably, the combination of 9.2.27PE and ABT-737 caused growth delay in a human melanoma xenograft mice model, supporting further investigations of this particular drug combination.

Malignant melanomas are generally drug resistant and have a very poor prognosis. We have studied the effects of a chemical conjugate of pseudomonas exotoxin A (PE) and the antibody 9.2.27, which recognizes the high molecular weight melanoma associated antigen (HMW-MAA) expressed in most malignant melanomas and melanoma cell lines. We demonstrate that the 9.2.27PE immunotoxin (IT) induces cell death in malignant melanoma cells through protein synthesis inhibition followed by some morphological and biochemical features of apoptosis, like rounding up of cells, chromatin condensation and inactivation of PARP. Unlike previous results with the 425.3PE IT in breast cancer cells, we detected no depolarization of the mitochondrial membrane after 9.2.27PE IT treatment. This is likely due to the lack of strong activation of caspase-8 and caspase-3. The lack of depolarization suggests that cytochrome c, a molecule that triggers activation of caspase-3, was retained within the mitochondria. In addition, the protein level of the antiapoptotic Bcl-2 did not decrease in contrast to other antiapoptotic molecules belonging to the inhibitor of apoptosis and the Bcl-2 family. This suggests that Bcl-2 may play a role in maintaining the mitochondrial membrane integrity in the 9.2.27PE-treated cells. Nevertheless, 9.2.27PE IT efficiently killed malignant melanoma cells that can be ascribed to inhibition of protein synthesis followed by some morphological and biochemical features of apoptosis. ' 2009 UICC Key words: immunotoxin; melanoma/skin cancer; effectors of apoptosis; antibodies Malignant melanoma is the major cause of death from skin cancer, and the incidence has increased significantly the past few decades. If diagnosed early, it can be cured by surgical resection. However, metastatic malignant melanomas are generally drug resistant and have a very poor prognosis.1 The response rate for dacarbazine (DTIC), a chemotherapeutic drug commonly used to treat metastatic malignant melanoma, is less than 20% and the complete response rate is below 5%.2 Hence, there is an obvious need for new therapies, preferably approaches that kill drug resistant cancer cells and not normal cells.Immunotoxins (ITs) are compounds that are being developed for cancer therapy. They consist of a toxin, derived from either plants or bacteria, which is linked to an antibody. The antibody recognizes cancer specific antigens. Once internalized, the IT induces cell death through 2 different mechanisms; inhibition of protein synthesis and induction of apoptosis. 3,4 We wanted to study the activity of the 9.2.27PE IT and to understand its mechanisms of action in melanoma cells.A complex interplay between pro-and antiapoptotic molecules ensures the homeostatic balance within the cell. A shift in this balance can lead to apoptosis. Apoptosis may occur via the extrinsic pathway (death-receptor pathway) or the intrinsic pathway (mitochondria-dependent). Both pathways involve activation of caspases, a group of proteolytic enzymes. Caspases are grouped into initiator c...

In human immunodeficiency virus type 1 (HIV-1) infection, functional activities of T lymphocytes are impaired. Analogous to tumor-infiltrating T lymphocytes from cancer patients, in whom poor proliferative responses are associated with fewer zeta molecules, this study compared expression of CD3zeta molecules by T lymphocytes from HIV-infected persons and healthy controls. Flow cytometry and immunoblotting revealed significantly diminished zeta expression by CD3, CD4, and CD8 T lymphocytes from AIDS patients but not from persons without AIDS. zeta-mRNA levels were also decreased in cells from AIDS patients. CD3zeta expression correlated significantly with CD4 cell counts and HIV-1 RNA levels; impaired expression of CD3zeta molecules appeared to be reversible upon virus load reduction following highly active antiretroviral treatment (HAART). Thus, reduced expression of CD3zeta molecules by T lymphocytes from HIV-infected persons correlates with disease status. Investigations into CD3zeta expression by subpopulations of peripheral T lymphocytes and by T lymphocytes in lymphoid tissues will contribute to the understanding of immune reconstitution of HIV-infected patients following HAART.

BackgroundTreatment of metastatic malignant melanoma patients harboring BRAF(V600E) has improved drastically after the discovery of the BRAF inhibitor, vemurafenib. However, drug resistance is a recurring problem, and prognoses are still very bad for patients harboring BRAF wild-type. Better markers for targeted therapy are therefore urgently needed.MethodologyIn this study, we assessed the individual kinase activity profiles in 26 tumor samples obtained from patients with metastatic malignant melanoma using peptide arrays with 144 kinase substrates. In addition, we studied the overall ex-vivo inhibitory effects of vemurafenib and sunitinib on kinase activity status.ResultsOverall kinase activity was significantly higher in lysates from melanoma tumors compared to normal skin tissue. Furthermore, ex-vivo incubation with both vemurafenib and sunitinib caused significant decrease in phosphorylation of kinase substrates, i.e kinase activity. While basal phosphorylation profiles were similar in BRAF wild-type and BRAF(V600E) tumors, analysis with ex-vivo vemurafenib treatment identified a subset of 40 kinase substrates showing stronger inhibition in BRAF(V600E) tumor lysates, distinguishing the BRAF wild-type and BRAF(V600E) tumors. Interestingly, a few BRAF wild-type tumors showed inhibition profiles similar to BRAF(V600E) tumors. The kinase inhibitory effect of vemurafenib was subsequently analyzed in cell lines harboring different BRAF mutational status with various vemurafenib sensitivity in-vitro.ConclusionsOur findings suggest that multiplex kinase substrate array analysis give valuable information about overall tumor kinase activity. Furthermore, intra-assay exposure to kinase inhibiting drugs may provide a useful tool to study mechanisms of resistance, as well as to identify predictive markers.

We have earlier shown that the 9.2.27 Pseudomonas Exotoxin A (PE) immunotoxin (IT) efficiently kills melanoma cells through inhibition of protein synthesis followed by some morphologic and biochemical features of apoptosis, a different cell killing mechanism than the one caused by Dacarbazine (DTIC), a chemotherapeutic drug used to treat malignant melanoma. To examine whether induced DTIC resistance also is a determining factor for the effectiveness of 9.2.27PE IT, we developed a DTIC resistant subline, FEMX-200DR, from the DTIC sensitive cell line FEMX. The cell variants were treated with 9.2.27PE, an IT binding to the high molecular weight-melanoma associated antigen (HMW-MAA) expressed on most malignant melanoma cells. The IT was equally effective in killing the FEMX-200DR and the FEMX cells, and the cell death was primarily caused by inhibition of protein synthesis. The DNA repair enzyme and apoptotic marker PARP, a substrate of caspase-3, was inactivated, although we observed only a minor activation of caspase-3 and caspase-8, intracellular proteases involved in apoptosis. In addition to being DTIC resistant, the FEMX-200DR cells were also more resistant to apoptosis than the parent cells as a 3 times higher concentration of the apoptotic inducer Staurosporine was needed to obtain IC50. Furthermore, in early passage malignant melanoma cell lines established from lymph node metastases, the 9.2.27PE caused a time-dependent and dose-dependent decrease in cell viability independent of their DTIC sensitivity. These findings show that the 9.2.27PE IT efficiently can cause cell death in malignant melanoma cells independent of their level of resistance to apoptosis and DTIC.