TMZ is an alkylating agent that causes cell death through DNA-damaging mechanisms.?To determine whether MMC(TMZ)-TOC induces the same cytotoxic effects through a receptor-dependent process, we first studied its DNA-damaging properties in IMR-32 cells that endogenously express SSTR2. We performed an alkaline comet assay to evaluate the ability of our drug conjugate to produce single- and double-stranded DNA breaks. As shown in?Figure 3A,B, the targeted agent-induced DNA breaks that were similar to free TMZ, resulting in migrating DNA fragments (comet tail) from the nucleoid (comet head). The damaging effects of MMC(TMZ)-TOC were significantly reduced (P?< 0.0001) when cells were preincubated with blocking doses of the high-affinity SSTR2 antagonist JR11, indicating receptor-dependent cytotoxicity. Next, we used a colony formation assay to assess the long-term survival and reproductive capability of the cells after treatment with the PDC. We treated BON1 (receptor-negative) and BON1-SSTR2 (receptor-positive) cells with 2 mu;mol/L MMC(TMZ)-TOC or 10 mu;mol/L TMZ for 5 consecutive days, seeded the cells at a low-density number, and allowed them to grow and form colonies for 14 days. The clonogenic survival data showed that MMC(TMZ)-TOC produced cytotoxic effects in the receptor-positive BON1-SSTR2 cells (P?< 0.01) similar to clinically relevant doses of TMZ. Conversely, no cytotoxicity was observed in receptor-negative BON1 cells following treatment with the drug conjugate, further illustrating the receptor-dependent effects of the PDC.

TMZ is an alkylating agent that causes cell death through DNA-damaging mechanisms.?To determine whether MMC(TMZ)-TOC induces the same cytotoxic effects through a receptor-dependent process, we first studied its DNA-damaging properties in IMR-32 cells that endogenously express SSTR2. We performed an alkaline comet assay to evaluate the ability of our drug conjugate to produce single- and double-stranded DNA breaks. As shown in?Figure 3A,B, the targeted agent-induced DNA breaks that were similar to free TMZ, resulting in migrating DNA fragments (comet tail) from the nucleoid (comet head). The damaging effects of MMC(TMZ)-TOC were significantly reduced (P?< 0.0001) when cells were preincubated with blocking doses of the high-affinity SSTR2 antagonist JR11, indicating receptor-dependent cytotoxicity. Next, we used a colony formation assay to assess the long-term survival and reproductive capability of the cells after treatment with the PDC. We treated BON1 (receptor-negative) and BON1-SSTR2 (receptor-positive) cells with 2 mu;mol/L MMC(TMZ)-TOC or 10 mu;mol/L TMZ for 5 consecutive days, seeded the cells at a low-density number, and allowed them to grow and form colonies for 14 days. The clonogenic survival data showed that MMC(TMZ)-TOC produced cytotoxic effects in the receptor-positive BON1-SSTR2 cells (P?< 0.01) similar to clinically relevant doses of TMZ. Conversely, no cytotoxicity was observed in receptor-negative BON1 cells following treatment with the drug conjugate, further illustrating the receptor-dependent effects of the PDC.

TMZ is an alkylating agent that causes cell death through DNA-damaging mechanisms. To determine whether MMC(TMZ)-TOC induces the same cytotoxic effects through a receptor-dependent process, we first studied its DNA-damaging properties in IMR-32 cells that endogenously express SSTR2. We performed an alkaline comet assay to evaluate the ability of our drug conjugate to produce single- and double-stranded DNA breaks. As shown in Figure 3A,B, the targeted agent-induced DNA breaks that were similar to free TMZ, resulting in migrating DNA fragments (comet tail) from the nucleoid (comet head). The damaging effects of MMC(TMZ)-TOC were significantly reduced (P < 0.0001) when cells were preincubated with blocking doses of the high-affinity SSTR2 antagonist JR11, indicating receptor-dependent cytotoxicity. Next, we used a colony formation assay to assess the long-term survival and reproductive capability of the cells after treatment with the PDC. We treated BON1 (receptor-negative) and BON1-SSTR2 (receptor-positive) cells with 2 umol/L MMC(TMZ)-TOC or 10 umol/L TMZ for 5 consecutive days, seeded the cells at a low-density number, and allowed them to grow and form colonies for 14 days. The clonogenic survival data showed that MMC(TMZ)-TOC produced cytotoxic effects in the receptor-positive BON1-SSTR2 cells (P < 0.01) similar to clinically relevant doses of TMZ. Conversely, no cytotoxicity was observed in receptor-negative BON1 cells following treatment with the drug conjugate, further illustrating the receptor-dependent effects of the PDC.

TMZ is an alkylating agent that causes cell death through DNA-damaging mechanisms. To determine whether MMC(TMZ)-TOC induces the same cytotoxic effects through a receptor-dependent process, we first studied its DNA-damaging properties in IMR-32 cells that endogenously express SSTR2. We performed an alkaline comet assay to evaluate the ability of our drug conjugate to produce single- and double-stranded DNA breaks. As shown in Figure 3A,B, the targeted agent-induced DNA breaks that were similar to free TMZ, resulting in migrating DNA fragments (comet tail) from the nucleoid (comet head). The damaging effects of MMC(TMZ)-TOC were significantly reduced (P < 0.0001) when cells were preincubated with blocking doses of the high-affinity SSTR2 antagonist JR11, indicating receptor-dependent cytotoxicity. Next, we used a colony formation assay to assess the long-term survival and reproductive capability of the cells after treatment with the PDC. We treated BON1 (receptor-negative) and BON1-SSTR2 (receptor-positive) cells with 2 umol/L MMC(TMZ)-TOC or 10 umol/L TMZ for 5 consecutive days, seeded the cells at a low-density number, and allowed them to grow and form colonies for 14 days. The clonogenic survival data showed that MMC(TMZ)-TOC produced cytotoxic effects in the receptor-positive BON1-SSTR2 cells (P < 0.01) similar to clinically relevant doses of TMZ. Conversely, no cytotoxicity was observed in receptor-negative BON1 cells following treatment with the drug conjugate, further illustrating the receptor-dependent effects of the PDC.