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Novel Pactamycin Analogs Induce p53 Dependent Cell-Cycle Arrest at S-Phase in Human Head and Neck Squamous Cell Carcinoma (HNSCC) Cells.

Guha G, Lu W, Li S, Liang X, Kulesz-Martin MF, Mahmud T, Indra AK, Ganguli-Indra G - PLoS ONE (2015)

Bottom Line: Furthermore, they do not induce apoptosis or autophagy in a dose- or a time-dependent manner, but induce mild senescence in the tested cell lines.Besides, the analogs mildly reduce cyclin D1 expression without affecting expression of cyclin B, Cdk2 and Cdk4.Specific inhibition of p53 by pifithrin-α reduces the percentage of cells accumulated in S-phase, suggesting contribution of p53 to S-phase increase.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, Oregon, United States of America.

ABSTRACT
Pactamycin, although putatively touted as a potent antitumor agent, has never been used as an anticancer drug due to its high cytotoxicity. In this study, we characterized the effects of two novel biosynthetically engineered analogs of pactamycin, de-6MSA-7-demethyl-7-deoxypactamycin (TM-025) and 7-demethyl-7-deoxypactamycin (TM-026), in head and neck squamous cell carcinoma (HNSCC) cell lines SCC25 and SCC104. Both TM-025 and TM-026 exert growth inhibitory effects on HNSCC cells by inhibiting cell proliferation. Interestingly, unlike their parent compound pactamycin, the analogs do not inhibit synthesis of nascent protein in a cell-based assay. Furthermore, they do not induce apoptosis or autophagy in a dose- or a time-dependent manner, but induce mild senescence in the tested cell lines. Cell cycle analysis demonstrated that both analogs significantly induce cell cycle arrest of the HNSCC cells at S-phase resulting in reduced accumulation of G2/M-phase cells. The pactamycin analogs induce expression of cell cycle regulatory proteins including master regulator p53, its downstream target p21Cip1/WAF1, p27kip21, p19, cyclin E, total and phospho Cdc2 (Tyr15) and Cdc25C. Besides, the analogs mildly reduce cyclin D1 expression without affecting expression of cyclin B, Cdk2 and Cdk4. Specific inhibition of p53 by pifithrin-α reduces the percentage of cells accumulated in S-phase, suggesting contribution of p53 to S-phase increase. Altogether, our results demonstrate that Pactamycin analogs TM-025 and TM-026 induce senescence and inhibit proliferation of HNSCC cells via accumulation in S-phase through possible contribution of p53. The two PCT analogs can be widely used as research tools for cell cycle inhibition studies in proliferating cancer cells with specific mechanisms of action.

No MeSH data available.


Related in: MedlinePlus

PCT analogs did not cause apoptosis or autophagy.(A): TUNEL assay: 24-h treatment with increasing concentrations (100 and 500 nM) of TM-025 and TM-026 did not show a significant (p<0.05) number of TUNEL+ apoptotic cells (green fluorescence) marked with arrow-heads in SCC25 and SCC104 cells. DNase I (100 ul; 10 U/ml; positive control) treated cells were used as a positive control. (B-C): Autophagy was estimated as a measure of ratio of cellular LC3-II to LC3-I. (B) Western blot depicting levels of LC3-I and LC3-II in PCT analog-treated cells. (C) The ratio of LC3-II:LC3-I did not show a significant (P<0.05) induction (represented by *) of autophagy in the PCT analog-treated cells, in comparison to the positive control (cells treated with 100 μl of 50 ng/ml rapamycin). All sample groups were found to be identical to the vehicle-treated group at the same level of significance (i.e., P<0.05).
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pone.0125322.g004: PCT analogs did not cause apoptosis or autophagy.(A): TUNEL assay: 24-h treatment with increasing concentrations (100 and 500 nM) of TM-025 and TM-026 did not show a significant (p<0.05) number of TUNEL+ apoptotic cells (green fluorescence) marked with arrow-heads in SCC25 and SCC104 cells. DNase I (100 ul; 10 U/ml; positive control) treated cells were used as a positive control. (B-C): Autophagy was estimated as a measure of ratio of cellular LC3-II to LC3-I. (B) Western blot depicting levels of LC3-I and LC3-II in PCT analog-treated cells. (C) The ratio of LC3-II:LC3-I did not show a significant (P<0.05) induction (represented by *) of autophagy in the PCT analog-treated cells, in comparison to the positive control (cells treated with 100 μl of 50 ng/ml rapamycin). All sample groups were found to be identical to the vehicle-treated group at the same level of significance (i.e., P<0.05).

Mentions: In order to determine whether the PCT analogs induce cell death, besides inhibiting cell proliferation, both apoptosis and canonical autophagy assays were performed. Fluorometric TUNEL analysis with TM-025 and TM-026-treated SCC25 and SCC104 cells (100 and 500 nM; 24 h) did not reveal any significant percentage of TUNEL+ cells after 24 h of treatment, in comparison to the DNase I-treated positive control [51] (Fig 4A). Along the same line, western blot analyses of a relatively high dose (1 μM) of TM-025/TM-026-treated (for 72 h) SCC104 cells did not detect any cleavage of caspase-3 (S2 Fig Part A) or PARP (S2 Fig Part B), using specific antibodies against anti-cleaved caspase-3 and PARP, respectively. Hence, for the considered doses and treatment time, TM-025 and TM-026 did not induce apoptosis in either of the two HNSCC cell lines. Furthermore, occurrence of autophagy by TM-025 or TM-026 treatment in SCC104 cells was determined by detecting and comparing levels of LC3-I and LC3-II cytosolic proteins by Western blot analyses after drug treatment (Fig 4B). Rapamycin-treated cells were used as a positive control (Fig 4B). Neither of the PCT analogs induced any autophagy (up to 100 nM of treatment for 48 h), as illustrated by the LC3-II: LC3-I ratio (Fig 4C). Altogether, our results suggest that TM-025 and TM-026 do not reduce cell viability via apoptosis or autophagy.


Novel Pactamycin Analogs Induce p53 Dependent Cell-Cycle Arrest at S-Phase in Human Head and Neck Squamous Cell Carcinoma (HNSCC) Cells.

Guha G, Lu W, Li S, Liang X, Kulesz-Martin MF, Mahmud T, Indra AK, Ganguli-Indra G - PLoS ONE (2015)

PCT analogs did not cause apoptosis or autophagy.(A): TUNEL assay: 24-h treatment with increasing concentrations (100 and 500 nM) of TM-025 and TM-026 did not show a significant (p<0.05) number of TUNEL+ apoptotic cells (green fluorescence) marked with arrow-heads in SCC25 and SCC104 cells. DNase I (100 ul; 10 U/ml; positive control) treated cells were used as a positive control. (B-C): Autophagy was estimated as a measure of ratio of cellular LC3-II to LC3-I. (B) Western blot depicting levels of LC3-I and LC3-II in PCT analog-treated cells. (C) The ratio of LC3-II:LC3-I did not show a significant (P<0.05) induction (represented by *) of autophagy in the PCT analog-treated cells, in comparison to the positive control (cells treated with 100 μl of 50 ng/ml rapamycin). All sample groups were found to be identical to the vehicle-treated group at the same level of significance (i.e., P<0.05).
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Related In: Results  -  Collection

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pone.0125322.g004: PCT analogs did not cause apoptosis or autophagy.(A): TUNEL assay: 24-h treatment with increasing concentrations (100 and 500 nM) of TM-025 and TM-026 did not show a significant (p<0.05) number of TUNEL+ apoptotic cells (green fluorescence) marked with arrow-heads in SCC25 and SCC104 cells. DNase I (100 ul; 10 U/ml; positive control) treated cells were used as a positive control. (B-C): Autophagy was estimated as a measure of ratio of cellular LC3-II to LC3-I. (B) Western blot depicting levels of LC3-I and LC3-II in PCT analog-treated cells. (C) The ratio of LC3-II:LC3-I did not show a significant (P<0.05) induction (represented by *) of autophagy in the PCT analog-treated cells, in comparison to the positive control (cells treated with 100 μl of 50 ng/ml rapamycin). All sample groups were found to be identical to the vehicle-treated group at the same level of significance (i.e., P<0.05).
Mentions: In order to determine whether the PCT analogs induce cell death, besides inhibiting cell proliferation, both apoptosis and canonical autophagy assays were performed. Fluorometric TUNEL analysis with TM-025 and TM-026-treated SCC25 and SCC104 cells (100 and 500 nM; 24 h) did not reveal any significant percentage of TUNEL+ cells after 24 h of treatment, in comparison to the DNase I-treated positive control [51] (Fig 4A). Along the same line, western blot analyses of a relatively high dose (1 μM) of TM-025/TM-026-treated (for 72 h) SCC104 cells did not detect any cleavage of caspase-3 (S2 Fig Part A) or PARP (S2 Fig Part B), using specific antibodies against anti-cleaved caspase-3 and PARP, respectively. Hence, for the considered doses and treatment time, TM-025 and TM-026 did not induce apoptosis in either of the two HNSCC cell lines. Furthermore, occurrence of autophagy by TM-025 or TM-026 treatment in SCC104 cells was determined by detecting and comparing levels of LC3-I and LC3-II cytosolic proteins by Western blot analyses after drug treatment (Fig 4B). Rapamycin-treated cells were used as a positive control (Fig 4B). Neither of the PCT analogs induced any autophagy (up to 100 nM of treatment for 48 h), as illustrated by the LC3-II: LC3-I ratio (Fig 4C). Altogether, our results suggest that TM-025 and TM-026 do not reduce cell viability via apoptosis or autophagy.

Bottom Line: Furthermore, they do not induce apoptosis or autophagy in a dose- or a time-dependent manner, but induce mild senescence in the tested cell lines.Besides, the analogs mildly reduce cyclin D1 expression without affecting expression of cyclin B, Cdk2 and Cdk4.Specific inhibition of p53 by pifithrin-α reduces the percentage of cells accumulated in S-phase, suggesting contribution of p53 to S-phase increase.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, Oregon, United States of America.

ABSTRACT
Pactamycin, although putatively touted as a potent antitumor agent, has never been used as an anticancer drug due to its high cytotoxicity. In this study, we characterized the effects of two novel biosynthetically engineered analogs of pactamycin, de-6MSA-7-demethyl-7-deoxypactamycin (TM-025) and 7-demethyl-7-deoxypactamycin (TM-026), in head and neck squamous cell carcinoma (HNSCC) cell lines SCC25 and SCC104. Both TM-025 and TM-026 exert growth inhibitory effects on HNSCC cells by inhibiting cell proliferation. Interestingly, unlike their parent compound pactamycin, the analogs do not inhibit synthesis of nascent protein in a cell-based assay. Furthermore, they do not induce apoptosis or autophagy in a dose- or a time-dependent manner, but induce mild senescence in the tested cell lines. Cell cycle analysis demonstrated that both analogs significantly induce cell cycle arrest of the HNSCC cells at S-phase resulting in reduced accumulation of G2/M-phase cells. The pactamycin analogs induce expression of cell cycle regulatory proteins including master regulator p53, its downstream target p21Cip1/WAF1, p27kip21, p19, cyclin E, total and phospho Cdc2 (Tyr15) and Cdc25C. Besides, the analogs mildly reduce cyclin D1 expression without affecting expression of cyclin B, Cdk2 and Cdk4. Specific inhibition of p53 by pifithrin-α reduces the percentage of cells accumulated in S-phase, suggesting contribution of p53 to S-phase increase. Altogether, our results demonstrate that Pactamycin analogs TM-025 and TM-026 induce senescence and inhibit proliferation of HNSCC cells via accumulation in S-phase through possible contribution of p53. The two PCT analogs can be widely used as research tools for cell cycle inhibition studies in proliferating cancer cells with specific mechanisms of action.

No MeSH data available.


Related in: MedlinePlus