Figure 1.
Differential H2S-releasing manner of NaHS and GYY4137.
(A) H2S-releasing profile of NaHS, GYY4137 and ZYJ1122. H2S released from NaHS, and GYY4137 (400 µM) was determined in aliquots (100 µl) of medium withdrawn at timed intervals (up to 7 days) from cultured MCF-7 cells. Concentration of H2S amounts was assessed spectrophotometrically using N,N-dimethyl-p-phenylenediamine-dihydrochloride and results showed the H2S concentration (µM). H2S release from GYY4137 was significantly different (P<0.05) from T = 0 at all time points from 0.3 hour to 7 days. H2S release from NaHS was significantly different (P<0.05) from T = 0 at all time points up to 1.5 hours. No detectable H2S was released from ZYJ1122 (400 µM) under identical experimental conditons. Results show mean ± s.e. mean, n = 3. The chemical structures of GYY4137 and ZYJ1122 are shown in the inset. (B) Growth curve analyses of MCF-7, HL-60 and MV4-11 cells treated with NaHS, GYY4137 and ZYJ1122 (400 µM) over 5 days. Cell survival was determined by trypan blue staining. Results show cell growth as a percentage relative to NT cell numbers at day 5 and are mean ± s.e. mean, n = 3.
Figure 2.
GYY4137 but not NaHS significantly affected cancer cell survivability.
(A) The effect of treatment (5 days) of a range of cancer and non-cancer cells with NaHS, GYY4137 and ZYJ1122 (400 µM or 800 µM) as determined by trypan blue staining. Results show percentage of cell viability to non-treatment (NT) following incubation in the absence of drug treatment and are mean ± s.e. mean, n = 3, (#P<0.05; *P<0.01). (B) Concentration-response curves showing the effect of GYY4137 treatment for 5 days on survival of MCF-7, HL-60 and MV4-11 cells. Results show mean ± s.e. mean, n = 3. (C) Representative photographs showing clonogenic survival assays of MCF-7 cells following exposure (5 days, 200–600 µM) to either NaHS (top row), GYY4137 (middle row) and ZYJ1122 (bottom row). NT = non-treatment.
Figure 3.
GYY4137 induced sub-G1 population and apoptosis in MCF-7.
(A) Cell cycle analysis of MCF-7 cells after 5 days (NT and ZYJ1122) and 5 and 8 days (GYY4137) drug treatment. Insets show percentage distribution of cells in each cell cycle phase. Results shown are indicative of 3 individual experiments. NT: non-treatment. (B) Western blot analysis of apoptosis markers (α-cleaved-PARP, α-cleaved-caspase 9) of MCF-7 and IMR90 treated (5 days) with GYY4137 or ZYJ1122 (both 400 µM). The anti-cleaved caspase-9 antibody used detects the large fragment of caspase-9 following cleavage but does not recognize uncleaved procaspase-9. α-tubulin was used as a loading control. Results shown are indicative of 3 individual experiments.
Figure 4.
Effect of GYY4137 on tumor growth in vivo.
Changes in volume of established tumors in, (A) HL-60 xenograft mice (B) MV4–11 xenograft mice treated daily with either GYY4137 (100, 200 and 300 mg/kg, i.p.) or vehicle control. Treatment with GYY4137 significantly reduced the tumor volume in both animal models, in a dose-dependent manner. Results show changes of tumor volume in mean ± s.e. mean (n = 4–6, # P<0.05; * P<0.01).