Table 1.
Primer pairs for HSP70 isoform quantification by real-time PCR.
Figure 1.
HSC70 (HSPA8) is the most prominent HSP70 isoform in MCC cell lines.
Δ−CT levels of the known HSP70 isoforms were determined applying SybrGreen real time PCR (red: MCPyV-positive; blue: MCPyV-negative; black: controls).
Figure 2.
HSC70 protein expression in vitro and in vivo.
HSC70 expression was assayed by immunohistochemistry applied to MCC cell lines embedded in a bovine plasma/thrombin clot prior to formalin fixation (A) and FFPE MCC metastasis of a patient (B). The depicted scale bar measures 20 μm.
Figure 3.
Heterogeneous MAL3-101 sensitivity of MCPyV-positive and MCPyV-negative tumor cell lines.
Cell lines were seeded with 10000 cells in 96-well plates and incubated for 24 h before they were treated for 72 h with the indicated concentrations of MAL3-101. Cell viability was determined by the trypan blue exclusion assay (A, B). Given are mean values (+/- SD) of three independent experiments. FM88 is a melanoma cell line, Jurkat cells are derived from a T-cell leukemia, while all other cell lines have been established from primary or metastatic MCCs. The MCPyV status was determined by real time PCR and by immunohistochemistry for the MCPyV Large T antigen. MCPyV-positive cell lines are grouped in A and C, and MCPyV-negative in B and D. The dashed line indicates an arbitrary threshold (80% viability) allowing the discrimination between MAL3-101 sensitive and resistant cell lines.
Figure 4.
MAL3-101 induces apoptosis in cell lines with high HSC70 expression.
(A) DNA staining of MAL3-101 treated WaGa cells (relative HSC70 mRNA level compared to primary fibroblasts: 22.6) reveals a strong increase in the sub-G1 population upon 20 h MAL3-101 treatment. In contrast, treatment of MKL-1 cells (relative HSC70 mRNA level compared to primary fibroblasts: 3.5) did not result in induction of a sub-G1 population. (B) Annexin-V/7AAD staining demonstrates increased early apoptosis (quadrant 3) as well as increased cell death (quadrant 4) in WaGa cells caused by MAL3-101 treatment (17 μM).
Figure 5.
MAL3-101 sensitivity of MCC cell lines correlates with HSC70 expression levels.
The relative HSC70 mRNA expression levels (with the lowest value arbitrarily set to 1) of the investigated cell lines were blotted against viability following 72 h of treatment with 17 μM MAL3-101. Gaussian distribution of the data was confirmed by using the Shapiro-Wilk normality test. Spearman's correlation coefficient as statistical test was applied.
Figure 6.
MAL3-101 treatment in an MCC xenotransplantation model demonstrated induction of apoptosis and reduced tumor growth.
WaGa cells embedded in MatriGel were injected s.c. in NOD.CB17-Prkdcscid/NCrHsd mice. Intraperitoneal injection of MAL3-101 (40 mg/kg) was started (arrow) on day 17 when the tumor volume had reached approximately 100 mm3 and was repeated every second day. (A) Mean values (± SEM, N = 6) of the tumor volume are depicted in the graph. The p-value was calculated using the 2-way ANOVA statistical test. (B) Representative macroscopic photographs depict the respective tumors at day 38. (C) IHC for cleaved caspase III in FFPE tumors excised on day 38 indicating caspase III dependent apoptosis induction in the MAL3-101 treated group compared to control group. The depicted scale bar measures 20 μm.