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. 2023 Mar 15;13(3):976–991.

Figure 4.

Figure 4

Par-4 secreted by normal cells treated with CZT inhibits cancer cell viability and tumor growth. (A) CZT induces Par-4 secretion in mouse embryonic fibroblasts (MEFs) from Par-4+/+ mice but not from Par-4-/- or p53-/- mice. The indicated MEFs were treated with CZT (1 µM) or vehicle (V) for 24 h, and the CM was examined for Par-4 secretion by western blot analysis. (B) CZT by itself does not inhibit cell viability of lung cancer cells. Lung cancer cells were treated with CZT (1 µM) or vehicle (V) for 24 h, and cell viability was examined by resazurin assays. (C) CM from CZT treated Par-4+/+ MEFs, but not Par-4-/- MEFs, inhibits cancer cell viability. Lung cancer cells A549, H1650, H1975, and KP7B were pretreated with CZT or vehicle (V) for 24 h and washed three times. The cells were then treated with the CM from normal cells Par-4+/+ or Par-4-/- MEFs treated with CZT. Prior to treating the cancer cells with the CM, we pre-incubated the CM for 30 min with Par-4 antibody, GRP78 antibody or control IgG. After 24 h of treatment, the cells were subjected to resazurin assays. Mean + SD values are shown. *P < 0.001 by Student’s t test. (D) CZT treatment inhibits the growth of xenografts derived from lung tumor cells. NSG mice were injected s.c. with H1650 or H1975 cells. When the tumors grew to 30 mm3, the mice were administered CZT (25 mg/kg body weight) or vehicle (n=5 per group) once every day for 19 consecutive days by oral gavage. (E) Inhibition of lung cancer growth by CZT in mice is Par-4-dependent. C57BL/6 mice that were either Par-4+/+ (wild type) or Par-4-/- were injected s.c. with KP7B cells, and the tumors were examined for response to CZT or vehicle. (D, E) Tumor volumes were determined at the indicated time intervals. Mean + SD is shown. **P < 0.01 by ANOVA test for tumor volumes at corresponding days in the vehicle control and CZT treatment groups of mice. Plasma samples from the mice treated with vehicle (V) or CZT collected at the end of the experiments were subjected to western blot analysis for Par-4 or Coomassie blue staining for albumin.