Figures & data
Figure 1 (A) Baneh or Daraban tree with clay cup for collecting resin. (B) The handmade muddy cup that was used for collecting exudate (resin). (C) Chewing gum produced from the natural MGR.
Table 1 Primer sequences in one-step SYBR Green quantitative real-time PCR
Figure 2 Total phenolic and flavonoid contents of MGR from Pistacia atlantica subspecies kurdica. Each value is the average of relative SD of 3 analyses.
Figure 3 Increase in radical scavenging activity of MGR: (A) DPPH and (B) ABTS+. *Significant difference from control (P < 0.05).
Figure 4 (A) IC50 value of 15.34 ± 0.21, 11.52 ± 0.18, 8.11 ± 0.23, and 5.2 ± 0.8 μg/mL at 72 hours of treatment with MGR for bile duct cancer (cholangiocarcinoma) (KMBC), pancreatic carcinoma (PANC-1), gastric adenocarcinoma (CRL-1739), and colonic adenocarcinoma (COLO205) cells. (B) The normal human colon fibroblasts (CCD-18Co) did not show any adverse effect after treatment with various doses of MGR.
Figure 5 Confocal micrographs of AO and PI double-stained COLO205 cells. Cells were treated with IC50 of MGR in a time-dependent manner. (A) Untreated cells showed normal structure without prominent apoptosis and necrosis. (B) Early apoptosis features were seen after 24 hours representing intercalated AO (bright green) among the fragmented DNA. (C) BL and nuclear margination were noticeable after 48 hours of treatment (arrows). (D) Late apoptosis was seen after 72 hours of treatment (a positive staining of orange color represents the hallmark of late apoptosis).
Table 2 Flow cytometry analysis of COLO205 cells after treatment with MGR, for which the cells were stained with FITC-conjugated annexin-V and PI and incubated at 37°C for 24, 48, and 72 hours
Figure 6 Induction of apoptosis in colonic adenocarcinoma (COLO205) cells by MGR which was observed by staining with FITC-conjugated annexin V-FITC. (A1–C1) Untreated (control) COLO205 cells at 24, 48, and 72 hours. (A2–C2) COLO205 cells after treatment with MGR at 12, 24, and 48 hours. *Significant (P < 0.05) increase in apoptotic cells in the MGR-treated groups compared to untreated controls.
Figure 7 The cell cycle of colonic adenocarcinoma (COLO205) cells treated with MGR after staining with PI. (A1–C1) Untreated (control) COLO205 cells at 24, 48, and 72 hours. (A2–C2) COLO205 cells treated with resin at 12, 24, and 48 hours. G0/G1, G2/M, and S are cell cycle phases, and sub-G0/G1 is the apoptotic cell population.
Table 3 Flow cytometry analysis of COLO205 cells after treatment with MGR, for which the cells were stained with PI and incubated at 37°C for 24, 48, and 72 hours
Table 3 Flow cytometry analysis of COLO205 cells after treatment with MGR, for which the cells were stained with PI and incubated at 37°C for 24, 48, and 72 hours
Figure 8 Activities of caspase-3 and -9. The total cell lysates from COLO205 cells treated with MGR for 24, 48, and 72 hours were analyzed using caspase colorimetric protease assay kits. *Significant difference from control (P < 0.05).
Table 4 Spectrophotometric analysis of caspases in COLO205 cells after treatment with MGR for 24, 48, and 72 hours
Table 4 Spectrophotometric analysis of caspases in COLO205 cells after treatment with MGR for 24, 48, and 72 hours
Figure 9 mRNA expression levels of Bcl-2, Bax, and Cyt-c normalized to the transcription levels of β-actin and GAPDH. The qRT-PCR analysis was performed on COLO205 cells treated with 5.2 ± 0.8 μg/mL MGR. The experiment was done in triplicates, and data are expressed as mean ± SD. *Significant difference from control (P < 0.05).
Figure 10 Proposed model of MGR mechanism of action behind its antiproliferative and apoptosis effects against colon adenocarcinoma in vitro.
