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SC06, a novel small molecule compound, displays preclinical activity against multiple myeloma by disrupting the mTOR signaling pathway.

Han K, Xu X, Xu Z, Chen G, Zeng Y, Zhang Z, Cao B, Kong Y, Tang X, Mao X - Sci Rep (2015)

Bottom Line: Mechanistic studies revealed that SC06 selectively inhibited the mTOR signaling pathway but had no effects on other associated kinases, such as AKT, ERK, p38, c-Src and JNK.Further studies showed that SC06-decreased mTOR activation was associated with the downregulation of Raptor, a key component of the mTORC1 complex.Notably, expression of Raptor, phosphorylation of mTOR and phosphorylated 4E-BP1 was also decreased in the tumor tissues from SC06-treated mice, which was consistent with the cellular studies.

View Article: PubMed Central - PubMed

Affiliation: Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-psycho-diseases, Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou, China.

ABSTRACT
The mammalian target of rapamycin (mTOR) is extensively involved in multiple myeloma (MM) pathophysiology. In the present study, we reported a novel small molecule SC06 that induced MM cell apoptosis and delayed MM xenograft growth in vivo. Oral administration of SC06 to mice bearing human MM xenografts resulted in significant inhibition of tumor growth at doses that were well tolerated. Mechanistic studies revealed that SC06 selectively inhibited the mTOR signaling pathway but had no effects on other associated kinases, such as AKT, ERK, p38, c-Src and JNK. Further studies showed that SC06-decreased mTOR activation was associated with the downregulation of Raptor, a key component of the mTORC1 complex. SC06 also suppressed the phosphorylation of 4E-BP1 and P70S6K, two typical substrates in the mTORC1 signaling pathway. Notably, expression of Raptor, phosphorylation of mTOR and phosphorylated 4E-BP1 was also decreased in the tumor tissues from SC06-treated mice, which was consistent with the cellular studies. Therefore, given the potency and low toxicity, SC06 could be developed as a potential anti-MM drug candidate by disrupting the mTOR signaling.

No MeSH data available.


Related in: MedlinePlus

SC06 inhibits mTORC1 signaling pathway in MM cells.(a) MM cell lines (LP1, OPM2, JJN3, RPMI-8226) were treated with 20 μM of SC06 or vehicle for 24 hr. After incubation, cells were harvested and whole lysates were prepared for immunoblotting assay against indicated specific antibodies. (b) LP1, OPM2, and JJN3 cells were treated with SC06 at increased concentrations for 24 hr, followed by immunoblotting for specific proteins as indicated. (c) MM cells were starved overnight followed by SC06 (10 μM) or rapamycin (10 nM) or chloroquine (CHQ, 20 μM) for 2 hr, followed by IGF-1 (100 ng/mL) treatment for 15 min. Cell lysates were then prepared for immunoblotting assay against p-mTOR, mTOR, or GAPDH. (d) Starved MM cells were treated with SC06 or S14161 (10 μM) for 2 hr, followed by IGF-1 (100 ng/mL) treatment for 15 min. Cell lysates were then prepared for immunoblotting assay against indicated proteins. (e) Immunoblotting assay for p-mTOR at Ser2448, mTOR, Raptor, Rictor and GAPDH in MM cell lines (LP1, OPM2, JJN3, U266, RPMI-8226). HEK293 cells were used as a negative control.
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f3: SC06 inhibits mTORC1 signaling pathway in MM cells.(a) MM cell lines (LP1, OPM2, JJN3, RPMI-8226) were treated with 20 μM of SC06 or vehicle for 24 hr. After incubation, cells were harvested and whole lysates were prepared for immunoblotting assay against indicated specific antibodies. (b) LP1, OPM2, and JJN3 cells were treated with SC06 at increased concentrations for 24 hr, followed by immunoblotting for specific proteins as indicated. (c) MM cells were starved overnight followed by SC06 (10 μM) or rapamycin (10 nM) or chloroquine (CHQ, 20 μM) for 2 hr, followed by IGF-1 (100 ng/mL) treatment for 15 min. Cell lysates were then prepared for immunoblotting assay against p-mTOR, mTOR, or GAPDH. (d) Starved MM cells were treated with SC06 or S14161 (10 μM) for 2 hr, followed by IGF-1 (100 ng/mL) treatment for 15 min. Cell lysates were then prepared for immunoblotting assay against indicated proteins. (e) Immunoblotting assay for p-mTOR at Ser2448, mTOR, Raptor, Rictor and GAPDH in MM cell lines (LP1, OPM2, JJN3, U266, RPMI-8226). HEK293 cells were used as a negative control.

Mentions: Next we investigated the underlying mechanisms of SC06 in its anti-MM action. Because kinases are key targets of many anti-cancer drugs, we performed an immunoblotting assay to measure the effects of SC06 on various kinases. It turned out that SC06 potently suppressed the activation of mTOR in terms of phosphorylation in MM cells. As shown in Fig. 3a, SC06 suppressed mTOR phosphorylation at both Ser2448 and Ser2481 sites in all examined MM cell lines, along with decreased phosphorylation of P70S6K and 4E-BP1, two hallmark substrates of mTORC1. However, SC06 did not decrease the activation level of AKT, a major upstream modulator kinase of mTOR, or other mTOR-associated kinases such as ERK, p-38, c-Src, or JNK10111213 (Supplemental Fig. 1). Further studies demonstrated that SC06 inhibited mTOR and its downstream signals P70S6K and 4E-BP1 in a concentration-dependent manner (Fig. 3b). The effects of SC06 on mTOR were further confirmed in starved MM cells stimulated by IGF-1. IGF-1 significantly stimulated mTOR phosphorylation in 15 min, which was completely abolished by rapamycin, a specific inhibitor of mTOR. Similar to rapamycin, SC06 also attenuated mTOR activation in the presence of IGF-1 (Fig. 3c). However, chloroquine (CHQ), a lysosomal inhibitor, failed to inhibit mTOR phosphorylation. To exclude the effects of this inhibition was due to the PI3K/AKT signaling pathway, we next performed an experiment in which MM cells were treated with S141618, a proven PI3K/AKT inhibitor, or SC06. As shown in Fig. 3d, S14161 abolished activation of AKT in all cell lines. Consistent with this finding, mTOR activation was also abolished by SC06, but SC06 had no effects on AKT activation. These results thus suggested that SC06 inhibited mTOR independent of AKT activation. Because mTOR activation is modulated by major components in the mTOR complex, we next evaluated the expression levels of phosphorylated mTOR, mTOR, Rictor and Raptor in MM cells. The results showed that all these proteins were highly expressed in all MM cell lines examined. In contrast, although the non-MM cell line HEK293 derived from human embryonic kidney expressed a high level of mTOR, a low level of activated mTOR was only detected. This finding was probably due to a low expression of Raptor and or Rictor (Fig. 3e). This expression pattern was consistent with cell viability affected by SC06. As shown in Fig. 1b, HEK293 cells were resistant to SC06, which probably due to the lack of activated mTOR. Therefore these results suggested that SC06 inhibited mTOR signaling pathway.


SC06, a novel small molecule compound, displays preclinical activity against multiple myeloma by disrupting the mTOR signaling pathway.

Han K, Xu X, Xu Z, Chen G, Zeng Y, Zhang Z, Cao B, Kong Y, Tang X, Mao X - Sci Rep (2015)

SC06 inhibits mTORC1 signaling pathway in MM cells.(a) MM cell lines (LP1, OPM2, JJN3, RPMI-8226) were treated with 20 μM of SC06 or vehicle for 24 hr. After incubation, cells were harvested and whole lysates were prepared for immunoblotting assay against indicated specific antibodies. (b) LP1, OPM2, and JJN3 cells were treated with SC06 at increased concentrations for 24 hr, followed by immunoblotting for specific proteins as indicated. (c) MM cells were starved overnight followed by SC06 (10 μM) or rapamycin (10 nM) or chloroquine (CHQ, 20 μM) for 2 hr, followed by IGF-1 (100 ng/mL) treatment for 15 min. Cell lysates were then prepared for immunoblotting assay against p-mTOR, mTOR, or GAPDH. (d) Starved MM cells were treated with SC06 or S14161 (10 μM) for 2 hr, followed by IGF-1 (100 ng/mL) treatment for 15 min. Cell lysates were then prepared for immunoblotting assay against indicated proteins. (e) Immunoblotting assay for p-mTOR at Ser2448, mTOR, Raptor, Rictor and GAPDH in MM cell lines (LP1, OPM2, JJN3, U266, RPMI-8226). HEK293 cells were used as a negative control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4556980&req=5

f3: SC06 inhibits mTORC1 signaling pathway in MM cells.(a) MM cell lines (LP1, OPM2, JJN3, RPMI-8226) were treated with 20 μM of SC06 or vehicle for 24 hr. After incubation, cells were harvested and whole lysates were prepared for immunoblotting assay against indicated specific antibodies. (b) LP1, OPM2, and JJN3 cells were treated with SC06 at increased concentrations for 24 hr, followed by immunoblotting for specific proteins as indicated. (c) MM cells were starved overnight followed by SC06 (10 μM) or rapamycin (10 nM) or chloroquine (CHQ, 20 μM) for 2 hr, followed by IGF-1 (100 ng/mL) treatment for 15 min. Cell lysates were then prepared for immunoblotting assay against p-mTOR, mTOR, or GAPDH. (d) Starved MM cells were treated with SC06 or S14161 (10 μM) for 2 hr, followed by IGF-1 (100 ng/mL) treatment for 15 min. Cell lysates were then prepared for immunoblotting assay against indicated proteins. (e) Immunoblotting assay for p-mTOR at Ser2448, mTOR, Raptor, Rictor and GAPDH in MM cell lines (LP1, OPM2, JJN3, U266, RPMI-8226). HEK293 cells were used as a negative control.
Mentions: Next we investigated the underlying mechanisms of SC06 in its anti-MM action. Because kinases are key targets of many anti-cancer drugs, we performed an immunoblotting assay to measure the effects of SC06 on various kinases. It turned out that SC06 potently suppressed the activation of mTOR in terms of phosphorylation in MM cells. As shown in Fig. 3a, SC06 suppressed mTOR phosphorylation at both Ser2448 and Ser2481 sites in all examined MM cell lines, along with decreased phosphorylation of P70S6K and 4E-BP1, two hallmark substrates of mTORC1. However, SC06 did not decrease the activation level of AKT, a major upstream modulator kinase of mTOR, or other mTOR-associated kinases such as ERK, p-38, c-Src, or JNK10111213 (Supplemental Fig. 1). Further studies demonstrated that SC06 inhibited mTOR and its downstream signals P70S6K and 4E-BP1 in a concentration-dependent manner (Fig. 3b). The effects of SC06 on mTOR were further confirmed in starved MM cells stimulated by IGF-1. IGF-1 significantly stimulated mTOR phosphorylation in 15 min, which was completely abolished by rapamycin, a specific inhibitor of mTOR. Similar to rapamycin, SC06 also attenuated mTOR activation in the presence of IGF-1 (Fig. 3c). However, chloroquine (CHQ), a lysosomal inhibitor, failed to inhibit mTOR phosphorylation. To exclude the effects of this inhibition was due to the PI3K/AKT signaling pathway, we next performed an experiment in which MM cells were treated with S141618, a proven PI3K/AKT inhibitor, or SC06. As shown in Fig. 3d, S14161 abolished activation of AKT in all cell lines. Consistent with this finding, mTOR activation was also abolished by SC06, but SC06 had no effects on AKT activation. These results thus suggested that SC06 inhibited mTOR independent of AKT activation. Because mTOR activation is modulated by major components in the mTOR complex, we next evaluated the expression levels of phosphorylated mTOR, mTOR, Rictor and Raptor in MM cells. The results showed that all these proteins were highly expressed in all MM cell lines examined. In contrast, although the non-MM cell line HEK293 derived from human embryonic kidney expressed a high level of mTOR, a low level of activated mTOR was only detected. This finding was probably due to a low expression of Raptor and or Rictor (Fig. 3e). This expression pattern was consistent with cell viability affected by SC06. As shown in Fig. 1b, HEK293 cells were resistant to SC06, which probably due to the lack of activated mTOR. Therefore these results suggested that SC06 inhibited mTOR signaling pathway.

Bottom Line: Mechanistic studies revealed that SC06 selectively inhibited the mTOR signaling pathway but had no effects on other associated kinases, such as AKT, ERK, p38, c-Src and JNK.Further studies showed that SC06-decreased mTOR activation was associated with the downregulation of Raptor, a key component of the mTORC1 complex.Notably, expression of Raptor, phosphorylation of mTOR and phosphorylated 4E-BP1 was also decreased in the tumor tissues from SC06-treated mice, which was consistent with the cellular studies.

View Article: PubMed Central - PubMed

Affiliation: Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-psycho-diseases, Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou, China.

ABSTRACT
The mammalian target of rapamycin (mTOR) is extensively involved in multiple myeloma (MM) pathophysiology. In the present study, we reported a novel small molecule SC06 that induced MM cell apoptosis and delayed MM xenograft growth in vivo. Oral administration of SC06 to mice bearing human MM xenografts resulted in significant inhibition of tumor growth at doses that were well tolerated. Mechanistic studies revealed that SC06 selectively inhibited the mTOR signaling pathway but had no effects on other associated kinases, such as AKT, ERK, p38, c-Src and JNK. Further studies showed that SC06-decreased mTOR activation was associated with the downregulation of Raptor, a key component of the mTORC1 complex. SC06 also suppressed the phosphorylation of 4E-BP1 and P70S6K, two typical substrates in the mTORC1 signaling pathway. Notably, expression of Raptor, phosphorylation of mTOR and phosphorylated 4E-BP1 was also decreased in the tumor tissues from SC06-treated mice, which was consistent with the cellular studies. Therefore, given the potency and low toxicity, SC06 could be developed as a potential anti-MM drug candidate by disrupting the mTOR signaling.

No MeSH data available.


Related in: MedlinePlus