Tumor radiosensitization by monomethyl auristatin E: mechanism of action and targeted delivery - PubMed (original) (raw)

Tumor radiosensitization by monomethyl auristatin E: mechanism of action and targeted delivery

Lisa Buckel et al. Cancer Res. 2015.

Abstract

Intrinsic tumor resistance to radiotherapy limits the efficacy of ionizing radiation (IR). Sensitizing cancer cells specifically to IR would improve tumor control and decrease normal tissue toxicity. The development of tumor-targeting technologies allows for developing potent radiosensitizing drugs. We hypothesized that the anti-tubulin agent monomethyl auristatin E (MMAE), a component of a clinically approved antibody-directed conjugate, could function as a potent radiosensitizer and be selectively delivered to tumors using an activatable cell-penetrating peptide targeting matrix metalloproteinases and RGD-binding integrins (ACPP-cRGD-MMAE). We evaluated the ability of MMAE to radiosensitize both established cancer cells and a low-passage cultured human pancreatic tumor cell line using clonogenic and DNA damage assays. MMAE sensitized colorectal and pancreatic cancer cells to IR in a schedule- and dose-dependent manner, correlating with mitotic arrest. Radiosensitization was evidenced by decreased clonogenic survival and increased DNA double-strand breaks in irradiated cells treated with MMAE. MMAE in combination with IR resulted in increased DNA damage signaling and activation of CHK1. To test a therapeutic strategy of MMAE and IR, PANC-1 or HCT-116 murine tumor xenografts were treated with nontargeted free MMAE or tumor-targeted MMAE (ACPP-cRGD-MMAE). While free MMAE in combination with IR resulted in tumor growth delay, tumor-targeted ACPP-cRGD-MMAE with IR produced a more robust and significantly prolonged tumor regression in xenograft models. Our studies identify MMAE as a potent radiosensitizer. Importantly, MMAE radiosensitization can be localized to tumors by targeted activatable cell-penetrating peptides.

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Figures

Figure 1. MMAE has increased potency compared to paclitaxel in tumor cells

A) HCT-116 (top panel) and PANC-1 (bottom panel) cells were exposed to 0, 1, 2 and 5 nM of MMAE for 24 hours. Cells were collected, stained with PI and cell cycle analyzed by FACS. B, C) HCT-116 and PANC-1 tumor cells were exposed to dose range of MMAE or paclitaxel for 72 hours. Cell viability was normalized to vehicle treated cells and plotted as fractional survival ± SD. D) IC50 of MMAE and paclitaxel in HCT-116, PANC-1, and 779E cells. Data are plotted as mean IC50 ± SD from triplicates. *P=0.003, **P=0.014, ***P=0.028

Figure 2. MMAE increases IR induced DNA double strand breaks in a schedule and dose dependent manner

A) HCT-116 cells were treated with 5 nM MMAE for 2, 4, or 24 hours followed by 6 Gy. Accumulation of cyclins was assessed by immunoblotting at time of irradiation. Comet tail length was measured using neutral comet assay 15 minutes post IR. Data are plotted as mean comet tail length ± SEM with non-irradiated comet tail length subtracted. B, C) HCT-116 cells were treated with 0, 1, or 5 nM MMAE for 24 hrs and then irradiated with 6 Gy. Comet tail length was measured using neutral comet assay. Data are plotted as mean comet tail length ± SEM. Representative images from comet tail assay are shown for MMAE dose of 5 nM. *P<0.01, **P<0.0001

Figure 3. MMAE decreases clonogenic survival of irradiated tumor cells

A, B) HCT-116 and PANC-1 cells were exposed to varying concentrations of MMAE overnight followed by 6 Gy. Cell viability was normalized to vehicle treated, non-irradiated cells and plotted as fractional survival ± SD. C, D) Clonogenic survival assay to measure radiosensitization. HCT-116 and PANC-1 cells were treated with 5 and 2 nM MMAE and then irradiated. Data are plotted as mean surviving fraction ± SD. E, F) The effect of MMAE with 2 Gy on cell survival was measured by clonogenic survival. Survival was normalized to non-irradiated cells for each concentration of MMAE. Data are plotted as mean survival ± SD. *P<0.01, **P<0.0001

Figure 4. MMAE increases DNA damage response in irradiated tumor cells

A) HCT-116 cells were treated with MMAE for 24 hrs, irradiated and 24 hours later apoptosis measured. Staurosporine treated cells were used as a positive apoptosis control. B, C) HCT-116 cells were treated with MMAE for 24 hours prior to 6 Gy and were collected 2 hours later. Lysates were immunoblotted for activation of CHK1 (pS345) and CHK2 (pT68) or cells were fixed and analyzed by immunofluorescence for γH2Ax foci formation D). Representative images of γH2AX foci formation in PANC-1 treated cells (green). Nuclei were stained with DAPI (blue). *P<0.05

Figure 5. Activatable cell penetrating peptides are cleaved in irradiated tumor microenvironments

A) Orthotopic pancreatic adenocarcinoma PDX were harvested and zymography gels used to assess gelatinase activity, lysates. For each PDX, lysates were run in duplicate (lanes A and B). B-D) HCT-116 or PANC-1 tumor xenografts were grown in both the left and right hindlimbs of nude mice. The right tumor was irradiated with 6 Gy and the left tumor was shielded to block out >95% of the IR dose. B) Zymography gels were used to asses MMP activity in non-irradiated and irradiated tumors. C) β3 integrin expression by IHC in non-irradiated and irradiated PANC-1 tumors D) One day post IR, ratiometric ACPP was intravenously injected and Cy5:Cy7 emission ratio measured (pseudocolor scale at far right) by whole animal imaging with tumors in situ and after tumor excision.

Figure 6. ACPP-cRGD-MMAE in combination with IR significantly reduces tumor growth

HCT-116 or PANC-1 tumor xenografts were grown subcutaneously in athymic nude mice. A) ACPP-cRGD-MMAE localizes to tumor xenografts following IV administration. The right hindlimb tumor was irradiated (3 Gy) while the left sided tumor was shielded to block >95% of the delivered IR dose. Cy5 labeled ACPP-cRGD-MMAE was IV injected into tumor bearing mice and mice were imaged 6 hrs later with skin on (top) and skin removed (bottom). B) Mice with HCT-116 tumor xenografts were IV injected with vehicle or 6 nmoles of ACPP-cRGD-MMAE. Tumor xenografts were harvested the following day, paraffin embedded and stained for mitotic marker pS10 Histone H3. C) PANC-1 tumor xenografts bearing mice were IV injected with 6 nmoles of free MMAE or ACPP-cRGD-MMAE days 0 and 1. For IR treated tumor xenografts, 3 Gy was delivered on days 1 and 2. Tumors were measured twice a week. D) HCT-116 tumors were treated 6 Gy on day 0 and then 3 Gy on days 1 and 2. A dose of 7.5 nM ACPP-cRGD-MMAE was IV injected on both days 0 and 1, 6 hrs after IR. Tumors were measured every other day.

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Tumor radiosensitization by monomethyl auristatin E: mechanism of action and targeted delivery - PubMed (original) (raw)