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δ-Tocotrienol oxazine derivative antagonizes mammary tumor cell compensatory response to CoCl2-induced hypoxia.

Ananthula S, Parajuli P, Behery FA, Alayoubi AY, Nazzal S, El Sayed K, Sylvester PW - Biomed Res Int (2014)

Bottom Line: In response to low oxygen supply, cancer cells elevate production of HIF-1α, a hypoxia-inducible transcription factor that subsequently acts to stimulate blood vessel formation and promote survival.Treatment with 150 µM CoCl2 induced a hypoxic response in +SA mammary tumor cells as evidenced by a large increase in HIF-1α levels, and combined treatment with compound 44 attenuated this response.Additional in vivo studies showed that intralesional treatment with compound 44 in BALB/c mice bearing +SA mammary tumors significantly decreased the levels of HIF-1α, and this effect was associated with a corresponding decrease in Akt/mTOR signaling and activation of downstream targets p70S6 kinase and eIF-4E1.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmacy, University of Louisiana at Monroe, 700 University Avenue, Monroe, LA 71209-0470, USA.

ABSTRACT
In response to low oxygen supply, cancer cells elevate production of HIF-1α, a hypoxia-inducible transcription factor that subsequently acts to stimulate blood vessel formation and promote survival. Studies were conducted to determine the role of δ-tocotrienol and a semisynthetic δ-tocotrienol oxazine derivative, compound 44, on +SA mammary tumor cell hypoxic response. Treatment with 150 µM CoCl2 induced a hypoxic response in +SA mammary tumor cells as evidenced by a large increase in HIF-1α levels, and combined treatment with compound 44 attenuated this response. CoCl2-induced hypoxia was also associated with a large increase in Akt/mTOR signaling, activation of downstream targets p70S6K and eIF-4E1, and a significant increase in VEGF production, and combined treatment with compound 44 blocked this response. Additional in vivo studies showed that intralesional treatment with compound 44 in BALB/c mice bearing +SA mammary tumors significantly decreased the levels of HIF-1α, and this effect was associated with a corresponding decrease in Akt/mTOR signaling and activation of downstream targets p70S6 kinase and eIF-4E1. These findings demonstrate that treatment with the δ-tocotrienol oxazine derivative, compound 44, significantly attenuates +SA mammary tumor cell compensatory responses to hypoxia and suggests that this compound may provide benefit in the treatment of rapidly growing solid breast tumors.

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Effects of 150 µM CoCl2 (hypoxic, but not cytotoxic dose) alone and in combination with compound 44 (44), on mitogenic/survival signaling and hypoxic response marker protein levels in +SA mammary tumor cells. +SA cells were seeded at concentration of 1.5 × 106 in 100 mm culture dishes and allowed to attach overnight. The following day, cells were divided into groups and exposed to their respective treatments for a 24 hr incubation period. Afterwards, whole cell lysates were prepared for Western blot analysis for Akt, PI3K, phospho-Akt (p-Akt, Ser473), mTOR, phospho-mTOR (p-mTOR, Ser2448), HIF-1α, phospho-p70S6K (p-p70S6K, Ser424), phospho-eIF-4E1 (p-eIF-4E1, Ser209), and phospho-4E-BP1 (p-4E-BP1, Thr37). Scanning densitometric analysis was performed on all blots done in triplicate and the integrated optical density of each band was normalized with corresponding α-tubulin, as shown in the bar graphs below their respective Western blot image. Vertical bars indicate the normalized integrated optical density of bands visualized in each lane ± SEM. *P < 0.05 as compared to the hypoxic CoCl2-treated controls.
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fig5: Effects of 150 µM CoCl2 (hypoxic, but not cytotoxic dose) alone and in combination with compound 44 (44), on mitogenic/survival signaling and hypoxic response marker protein levels in +SA mammary tumor cells. +SA cells were seeded at concentration of 1.5 × 106 in 100 mm culture dishes and allowed to attach overnight. The following day, cells were divided into groups and exposed to their respective treatments for a 24 hr incubation period. Afterwards, whole cell lysates were prepared for Western blot analysis for Akt, PI3K, phospho-Akt (p-Akt, Ser473), mTOR, phospho-mTOR (p-mTOR, Ser2448), HIF-1α, phospho-p70S6K (p-p70S6K, Ser424), phospho-eIF-4E1 (p-eIF-4E1, Ser209), and phospho-4E-BP1 (p-4E-BP1, Thr37). Scanning densitometric analysis was performed on all blots done in triplicate and the integrated optical density of each band was normalized with corresponding α-tubulin, as shown in the bar graphs below their respective Western blot image. Vertical bars indicate the normalized integrated optical density of bands visualized in each lane ± SEM. *P < 0.05 as compared to the hypoxic CoCl2-treated controls.

Mentions: Treatment for 24 hr to 150 µM CoCl2 alone had little or no effect on total levels of Akt, PI3K, and mTOR or phospho-Akt but did cause a relatively large increase in phospho-mTOR, HIF-1α, phospho-p70S6K, phospho-elF-4E1 levels in +SA mammary tumor cells, as compared to the vehicle-treated control group (Figure 5). Treatment with 2 μM compound 44 alone had little or no effect on total Akt, mTOR, HIF-1α, phospho-p70S6K, or phospho-eIF-4E1 levels but caused a relatively large decrease in total PI3K, phospho-Akt, and phospho-mTOR and corresponding increase in phospho-4E-BP1 levels in +SA cells as compared to the vehicle-treated control group (Figure 5). Combined treatment with these same doses of CoCl2 and compound 44 attenuated the hypoxic response characterized by a blockade of CoCl2-induced increases in phospho-mTOR, HIF-1α, phospho-p70S6K, and phospho-eIF-4E1 (Figure 5). In addition, phospho-4E-BP1 levels remained significantly increased in the combined treatment group as compared to the group treated with CoCl2 alone (Figure 5).


δ-Tocotrienol oxazine derivative antagonizes mammary tumor cell compensatory response to CoCl2-induced hypoxia.

Ananthula S, Parajuli P, Behery FA, Alayoubi AY, Nazzal S, El Sayed K, Sylvester PW - Biomed Res Int (2014)

Effects of 150 µM CoCl2 (hypoxic, but not cytotoxic dose) alone and in combination with compound 44 (44), on mitogenic/survival signaling and hypoxic response marker protein levels in +SA mammary tumor cells. +SA cells were seeded at concentration of 1.5 × 106 in 100 mm culture dishes and allowed to attach overnight. The following day, cells were divided into groups and exposed to their respective treatments for a 24 hr incubation period. Afterwards, whole cell lysates were prepared for Western blot analysis for Akt, PI3K, phospho-Akt (p-Akt, Ser473), mTOR, phospho-mTOR (p-mTOR, Ser2448), HIF-1α, phospho-p70S6K (p-p70S6K, Ser424), phospho-eIF-4E1 (p-eIF-4E1, Ser209), and phospho-4E-BP1 (p-4E-BP1, Thr37). Scanning densitometric analysis was performed on all blots done in triplicate and the integrated optical density of each band was normalized with corresponding α-tubulin, as shown in the bar graphs below their respective Western blot image. Vertical bars indicate the normalized integrated optical density of bands visualized in each lane ± SEM. *P < 0.05 as compared to the hypoxic CoCl2-treated controls.
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fig5: Effects of 150 µM CoCl2 (hypoxic, but not cytotoxic dose) alone and in combination with compound 44 (44), on mitogenic/survival signaling and hypoxic response marker protein levels in +SA mammary tumor cells. +SA cells were seeded at concentration of 1.5 × 106 in 100 mm culture dishes and allowed to attach overnight. The following day, cells were divided into groups and exposed to their respective treatments for a 24 hr incubation period. Afterwards, whole cell lysates were prepared for Western blot analysis for Akt, PI3K, phospho-Akt (p-Akt, Ser473), mTOR, phospho-mTOR (p-mTOR, Ser2448), HIF-1α, phospho-p70S6K (p-p70S6K, Ser424), phospho-eIF-4E1 (p-eIF-4E1, Ser209), and phospho-4E-BP1 (p-4E-BP1, Thr37). Scanning densitometric analysis was performed on all blots done in triplicate and the integrated optical density of each band was normalized with corresponding α-tubulin, as shown in the bar graphs below their respective Western blot image. Vertical bars indicate the normalized integrated optical density of bands visualized in each lane ± SEM. *P < 0.05 as compared to the hypoxic CoCl2-treated controls.
Mentions: Treatment for 24 hr to 150 µM CoCl2 alone had little or no effect on total levels of Akt, PI3K, and mTOR or phospho-Akt but did cause a relatively large increase in phospho-mTOR, HIF-1α, phospho-p70S6K, phospho-elF-4E1 levels in +SA mammary tumor cells, as compared to the vehicle-treated control group (Figure 5). Treatment with 2 μM compound 44 alone had little or no effect on total Akt, mTOR, HIF-1α, phospho-p70S6K, or phospho-eIF-4E1 levels but caused a relatively large decrease in total PI3K, phospho-Akt, and phospho-mTOR and corresponding increase in phospho-4E-BP1 levels in +SA cells as compared to the vehicle-treated control group (Figure 5). Combined treatment with these same doses of CoCl2 and compound 44 attenuated the hypoxic response characterized by a blockade of CoCl2-induced increases in phospho-mTOR, HIF-1α, phospho-p70S6K, and phospho-eIF-4E1 (Figure 5). In addition, phospho-4E-BP1 levels remained significantly increased in the combined treatment group as compared to the group treated with CoCl2 alone (Figure 5).

Bottom Line: In response to low oxygen supply, cancer cells elevate production of HIF-1α, a hypoxia-inducible transcription factor that subsequently acts to stimulate blood vessel formation and promote survival.Treatment with 150 µM CoCl2 induced a hypoxic response in +SA mammary tumor cells as evidenced by a large increase in HIF-1α levels, and combined treatment with compound 44 attenuated this response.Additional in vivo studies showed that intralesional treatment with compound 44 in BALB/c mice bearing +SA mammary tumors significantly decreased the levels of HIF-1α, and this effect was associated with a corresponding decrease in Akt/mTOR signaling and activation of downstream targets p70S6 kinase and eIF-4E1.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmacy, University of Louisiana at Monroe, 700 University Avenue, Monroe, LA 71209-0470, USA.

ABSTRACT
In response to low oxygen supply, cancer cells elevate production of HIF-1α, a hypoxia-inducible transcription factor that subsequently acts to stimulate blood vessel formation and promote survival. Studies were conducted to determine the role of δ-tocotrienol and a semisynthetic δ-tocotrienol oxazine derivative, compound 44, on +SA mammary tumor cell hypoxic response. Treatment with 150 µM CoCl2 induced a hypoxic response in +SA mammary tumor cells as evidenced by a large increase in HIF-1α levels, and combined treatment with compound 44 attenuated this response. CoCl2-induced hypoxia was also associated with a large increase in Akt/mTOR signaling, activation of downstream targets p70S6K and eIF-4E1, and a significant increase in VEGF production, and combined treatment with compound 44 blocked this response. Additional in vivo studies showed that intralesional treatment with compound 44 in BALB/c mice bearing +SA mammary tumors significantly decreased the levels of HIF-1α, and this effect was associated with a corresponding decrease in Akt/mTOR signaling and activation of downstream targets p70S6 kinase and eIF-4E1. These findings demonstrate that treatment with the δ-tocotrienol oxazine derivative, compound 44, significantly attenuates +SA mammary tumor cell compensatory responses to hypoxia and suggests that this compound may provide benefit in the treatment of rapidly growing solid breast tumors.

Show MeSH
Related in: MedlinePlus