Modulation of STAT3 Signaling, Cell Redox Defenses and Cell Cycle Checkpoints by β-Caryophyllene in Cholangiocarcinoma Cells: Possible Mechanisms Accounting for Doxorubicin Chemosensitization and Chemoprevention

Abstract

Cholangiocarcinoma (CCA) is an aggressive group of biliary tract cancers, characterized by late diagnosis, low effective chemotherapies, multidrug resistance, and poor outcomes. In the attempt to identify new therapeutic strategies for CCA, we studied the antiproliferative activity of a combination between doxorubicin and the natural sesquiterpene β-caryophyllene in cholangiocarcinoma Mz-ChA-1 cells and nonmalignant H69 cholangiocytes, under both long-term and metronomic schedules. The modulation of STAT3 signaling, oxidative stress, DNA damage response, cell cycle progression and apoptosis was investigated as possible mechanisms of action. β-caryophyllene was able to synergize the cytotoxicity of low dose doxorubicin in Mz-ChA-1 cells, while producing cytoprotective effects in H69 cholangiocytes, mainly after a long-term exposure of 24 h. The mechanistic analysis highlighted that the sesquiterpene induced a cell cycle arrest in G2/M phase along with the doxorubicin-induced accumulation in S phase, reduced the γH2AX and GSH levels without affecting GSSG. ROS amount was partly lowered by the combination in Mz-ChA-1 cells, while increased in H69 cells. A lowered expression of doxorubicin-induced STAT3 activation was found in the presence of β-caryophyllene in both cancer and normal cholangiocytes. These networking effects resulted in an increased apoptosis rate in Mz-ChA-1 cells, despite a lowering in H69 cholangiocytes. This evidence highlighted a possible role of STAT3 as a final effector of a complex network regulated by β-caryophyllene, which leads to an enhanced doxorubicin-sensitivity of cholangiocarcinoma cells and a lowered chemotherapy toxicity in nonmalignant cholangiocytes, thus strengthening the interest for this natural sesquiterpene as a dual-acting chemosensitizing and chemopreventive agent.

Chemopreventive Potential of Caryophyllane Sesquiterpenes: An Overview of Preliminary Evidence

“…This effect was found associated with an increased cell cycle arrest in G2/M phase, thus suggesting that as a consequence of the increased γH2AX by doxorubicin, β-caryophyllene stimulates G2/M checkpoint and DNA repair systems in order to block the cell cycle and enable restoring the genome integrity. This hypothesis is also supported by the evidence of a lower genoprotective effect in cholangiocarcinoma cells, which is likely due to the presence of defective DNA repair systems, as found in different cancer cells [ 163 ].…”

“…Indeed, it has been characterized by a plethora of biological activities, among which analgesic, anti-inflammatory, antioxidant, neuroprotective, and antiproliferative were the most investigated; moreover, it has been reported to affect phospholipid cooperativity and membrane permeability ( Table 3 ). These properties have provided benefits in several experimental models of disease, such as neurodegeneration, inflammation, pain, anxiety, depression, autoimmune diseases, metabolic ailments, osteoarthritis and some cancer models [ 123 , 124 , 125 , 126 , 127 , 128 , 129 , 130 , 131 , 132 , 133 , 134 , 135 , 136 , 137 , 138 , 139 , 140 , 141 , 142 , 143 , 144 , 145 , 146 , 147 , 148 , 149 , 150 , 151 , 152 , 153 , 154 , 155 , 156 , 157 , 158 , 159 , 160 , 161 , 162 , 163 , 164 , 165 , 166 , 167 , 168 ,…”

“…Caryophyllane sesquiterpenes from the plant kingdom were studied for their ability to counteract DNA damage induced by different toxicants, including environmental pollutants, such as cigarette smoke and butts, carcinogens, and drugs [ 163 , 171 , 172 , 173 , 174 , 175 , 176 ]. Particularly, in the Salmonella reverse mutation assay (pre-incubation method), β-caryophyllene strongly inhibited the mutagenicity of 2-nitrofluorene in Salmonella typhimurium TA98 strain [ 171 ].…”

“…Recently, we have highlighted the ability of β-caryophyllene to inhibit the genotoxic damage of the anticancer drug doxorubicin in cholangiocarcinoma cells and especially in noncancerous cholangiocytes as evidenced by the lowering in the levels of phosphorylated (Ser139) histone 2AX (namely γH2AX), which is an early biomarker of DNA double-strand breaks [ 163 ]. This effect was found associated with an increased cell cycle arrest in G2/M phase, thus suggesting that as a consequence of the increased γH2AX by doxorubicin, β-caryophyllene stimulates G2/M checkpoint and DNA repair systems in order to block the cell cycle and enable restoring the genome integrity.…”

Genipin Suppresses Growth and Metastasis in Hepatocellular Carcinoma Through Blocking Activation of STAT-3

“…al. have revealed that STAT-3 was constitutively activated in malignancy instead of normal tissues [35,36]. Interestingly, our results showed that genipin selectively suppressed HCC cells proliferation without signi cant toxicity in normal cells.…”