The hypoxia status of the BM and the in vitro effects of TH-302 on MM cells. (A) Immunohistochemistry staining of exogenous hypoxia marker and endogenous hypoxia marker in BM sections of naive and 5T33MM mice. Hypoxia was determined by the accumulation of pimonidazole and HIF-1α as described in supplemental Methods. Original magnification, × 40. Representative pictures, n = 6/group. (B) TH-302 induces G0/G1 cell-cycle arrest in 5T33vt cells in a hypoxia-selective manner. Similar results were also found in RPMI-8226, LP-1, MMS1, and Karpas-707 MM cells (data not shown). (C) Effects of TH-302 on components of the 5T33vt MM cell-cycle machinery. TH-302-induced G0/G1 cell-cycle arrest depends on down-regulating cyclin D1/2/3, CDK4/6, p21, p27, and pRb expression. Similar results were also found in RPMI-8226, LP-1, MMS1, and Karpas-707 MM cells (data not shown). (D) TH-302 triggers specific apoptosis in a dose-dependent manner in LP-1 cells under hypoxia. *P < .05, **P < .01, ***P < .001, compared with 20% O2 (n = 3). Similar results were also seen with RPMI-8226, 5T33vt, MMS1, and Karpas-707 cells (data not shown). (E) The mechanism of TH-302-induced apoptosis in LP-1 cells. Similar results were also found in RPMI-8226, 5T33vt, MMS1, and Karpas-707 cells. TH-302 = 5μM. (F) TH-302 decreases the accumulation of HIF-1α in hypoxic RPMI-8226 cells. Similar results were also found in 5T33vt, LP-1, MMS1, and Karpas-707 cells (data not shown). TH-302 = 5μM. (G) VEGFa secretion was reduced by TH-302 in 5T33vt cells. *P < .05. n = 3.
