Cortex periplocae is the dried root bark of
Liver cancer is the fifth leading malignancy in men and the ninth in women worldwide [
Current treatments for liver cancer are limited. Hepatectomy can be used in early stage liver cancer patients with functional liver. Liver transplantation can help patients with damaged livers, but matching suitable donors is not easy. Although these surgical operations work well in early stage diseases, they are not helpful for patients with cancer cells spread out of the liver. Chemotherapy and internal radiation therapy are also options for liver cancer treatment. However, they may do damage to other tissues and organs as well. Targeted therapy is a more specific treatment for cancer. Sorafenib, a multireceptor kinase inhibitor with antiangiogenic activity, is the standard treatment for advanced hepatocellular carcinoma (HCC) that cannot be removed with surgery. Although it extends the median overall survival in patients with advanced HCC for nearly 3 months, sorafenib does not increase the median time to symptomatic progression in patients with advanced HCC [
With the advancement of techniques in extraction, isolation, and recognition of compounds from plants, scientists started to search for antitumor components from herb medicine [
TNF-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor superfamily. It is also known as CD253 and APO-2L. TRAIL binds to the death receptors DR4 and DR5 and induces cell apoptosis [
Although TRAIL is a promising chemotherapeutic target for cancers, resistance to TRAIL-induced apoptosis has been reported in several different cancers, including colorectal cancer, breast cancer, liver cancer, and pancreatic cancer [
HCC cell lines were purchased from different organizations. HA22T/VGH and Huh-7 were purchased from Bioresource Collection and Research Center (BCRC) in Taiwan. Huh-7 was purchased from Japanese Collection of Research Bioresources (JCRB). HA22T/VGH and Huh-7 were culture in DMEM (Gibco, Carlsbad, CA, USA) with 10% FBS and 100 mM nonessential amino acids (Gibco, Carlsbad, CA, USA).
Recombinant human soluble TRAIL/APO2 ligand was purchased from ProSpec (Tany TechnoGene Ltd., Israel). Z-DEVD-FMK (CASP3 inhibitor), Z-IETD-FMK (CASP8 inhibitor), Z-LEHD-FMK (CASP9 inhibitor), and Z-VAD-FMK (pan CASP inhibitor) were purchased from R and D (Minneapolis, MN, USA). Monoclonal antihuman TRAIL R1 (TNFRSF10A,DR4)-Phycoerythrin antibody, antihuman TRAIL R3 (TNFRSF10C, DcR1)-Phycoerythrin antibody, and antihuman TRAIL R4 (TNFRSF10D, DcR2)-Phycoerythrin antibody were purchased from R and D (Minneapolis, MN, USA). PE antihuman TRAIL-R2 (TNFRSF10B, DR5) antibody was purchased from Biolegend. (San Diego, CA, USA) N-acetyl-cysteine (NAC) and DCHFDA were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Hydrogen peroxide (H2O2) was purchased from MERCK (Whitehouse Station, NJ, USA).
Total cellular lysates were prepared by using RIPA lysis buffer. Proteins in cell lysates (50
Cells were seeded at 104 cells in 100 uL medium per well in a 96-well plate and incubated (37°C, 5% CO2) overnight. Drugs of interest were added to each well and incubated (37°C, 5% CO2) for 2 days. MTT solution (5 mg/mL, Sigma Chemical Co., St. Louis, MO, USA) was added to each well at a final concentration of 0.5 mg/mL and incubated (37°C, 5% CO2) for 1-2 hours. Afterward, 100 uL of 10% SDS (Fluka, St. Louis, MO, USA) was added to each well and incubated at room temperature overnight.
Optical density at 570 nm was detected by SpectraMax M5 (Molecular Devices, Sunnyvale, CA, USA) for quantification.
ROS production was monitored by flow cytometry using DCFH-DA. This dye is a stable compound that readily diffuses into cells and is hydrolyzed by intracellular esterase to yield DCFH, which is trapped within cells. Thus, the fluorescence intensity is detected to quantify the amount of peroxide produced by the cells. To investigate the effect of periplocin and the combination of periplocin and TRAIL on generating intracellular ROS in HA22T/VGH, cells were pretreated with N-acetyl-cysteine (NAC) (30 mM) for 30 min and followed by periplocin (0.3, 0.03
After treated with periplocin alone or together with TRAIL for 24 hours, HA22T/VGH cells were washed and resuspended in the staining buffer and examined with the Vybrant Apoptosis Assay Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The cell suspension was incubated with 2.5
Trypsinized cells were washed with ice-cold PBS and fixed in 70% ethanol at −20°C for at least 1 h. After fixation, cells were washed twice, incubated in 0.5 mL of 0.5% Triton X-100/PBS at 37°C for 30 min with 1 mg/mL of RNase A, and stained with 0.5 mL of 50 mg/mL propidium iodide for 10 min. The fluorescence emitted from the propidium-DNA complex was quantitated by FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA).
Cells were incubated with dye-labeled monoclonal antibodies (mAb) against target molecules for 30 min on ice. Stained cells were then washed twice and resuspended in cold buffer and analyzed with a FACScan flow cytometry (BD Biosciences, San Jose, CA, USA). More than
All experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC number: ITRI-IACUC-2012-010M, Industrial Technology Research Institute of Taiwan, HsinChu, Taiwan. SCID (CB17/Icr-Prkdcscid/CrlBltw) mice were purchased from BioLASCO Ltd. (Ilan, Taiwan). Huh-7 cells (3 × 106 cells per mice) in 100
Huh-7 tumors were allowed to grow to 100–200 mm3. Periplocin (5–20 mg/kg;
At the end of the study, mice were sacrificed, and tumor samples were collected, fixed in formalin, and embedded in paraffin as tissue sections. Tissue sections were stained with hematoxylin and eosin (H and E) for general tissue morphology evaluation. The antihuman Ki67 antibody (1 : 500 dilution, IS-626, Dako, Glostrup Denmark) and antihuman cyclin-D1 antibody (1 : 500 dilution, IS-626, Dako, Glostrup, Denmark) were used in immunohistochemistry staining. Staining procedure was completed by using Autostainer Link 48 system (Dako, Glostrup Denmark). Five fields of every tumor sample were randomly selected, and the percentage of the Ki67-positive and cyclin-D1-positive cells was calculated to evaluate the proliferation of tumor samples.
Cortex periplocae (CP) is a traditional medicine capable of inhibiting cancer cell growth. To identify the active ingredients in CP that are responsible for its activity in inhibiting the growth of hepatocellular carcinoma (HCC) cells, we isolated and purified CP. After several rounds of purification, we found a group of compounds named CP-1 to 6 as major components in the fraction that can actively inhibit tumor cell growth (Figure
The effect of cortex periplocae extracts on HCC cell viability. (a) The chemical structures and names of purified cortex periplocae extracts are listed. (b) The cell viability of HA22T/VGH cells was determined by MTT assay after cells were treated with different concentrations of the indicated compound (0.03, 0.1, 0.3, 1, 3, and 10
To further identify the active ingredients in the fraction, the pure compounds in the active fraction was examined one by one, and periplocin (CP-1) was identified as the most potent compound in inhibiting tumor cell growth with IC50 at 0.027
Although TRAIL is a promising anticancer drug, more and more TRAIL-resistant cancers were reported. We sought to determine if periplocin can sensitize TRAIL-resistant HCC cells to TRAIL treatment. As shown in Figure
Periplocin and TRAIL treatments regulated cell apoptosis in HA22T/VGH cells. (a) HA22T/VGH cells were treated with periplocin alone or together with TRAIL for 24 h, and the morphology of cells was observed under microscope. (b) Cell apoptosis in HA22T/VGH cells regulated by periplocin and/or TRAIL was determined by FACS with Annexin-V-Fluorescein (
To study if the combination treatments of periplocin and TRAIL sensitize TRAIL-resistant HCC cells and induce HCC apoptosis, HA22T/VGH cells were stained with Annexin V and PI to characterize cells in early and late stages of apoptotic processes accordingly. As shown in Figure
Moreover, the accumulation of cell debris after apoptosis was demonstrated by sub-G1 population in cell cycle analysis. Consistent with our previous results, periplocin dose dependently increased sub-G1 population in HCC cells, while the addition of TRAIL further increased the sub-G1 population in HCC cells (Figure
Intracellular reactive oxygen species (ROS) are involved in apoptotic pathways. Therefore, the effects of periplocin and/or TRAIL treatments on the intracellular ROS level in HA22T/VGH cells were examined. Although periplocin treatment alone or together with TRAIL induced intracellular ROS accumulation in HA22T/VGH cells, NAC pretreatment did not prevent cell apoptosis induced by TRAIL and periplocin cotreatment (supplemental Figure 1 (see Supplementary Material available online at http://dx.doi.org/10.1155/2013/958025.) and data not shown)
TRAIL induces cell apoptosis through interaction with death receptors DR4 and DR5 signaling. It was reported that compounds which upregulate DR4 and DR5 in HCC cells could sensitize TRAIL-resistant HCC cells to TRAIL treatment [
Treatments of periplocin and/or TRAIL activate DR4, FADD, and proapoptotic proteins in HCC cells. (a) The effect of periplocin treatment on the expression of DR4, DR5, and FADD was analyzed by western blot. (b) HA22T/VGH cells were treated with different doses of the indicated compounds for 24 h. Expressions of both proforms and cleaved forms of caspase-8, caspase 3, PARP, and BID were analyzed by western blotting. (c) Huh-7 cells were treated with different concentrations of the indicated compounds for 4 h or 24 h. The expression of caspase-8, caspase-9 was analyzed after indicated compounds treatment for 4 h, and the expression of caspase 3, PARP, BID was analyzed after the treatment of indicated compounds for 24 h. (d) HA22T/VGH cells were pretreated with 20
TRAIL binds DR4 and activates FADD. Activated FADD induces the cleavage of several proapoptotic proteins and activates them. We examined the activation of several apoptosis-related proteins, including the cleavage of BID, caspase 8, caspase 3, and PARP in HA22T/VGH. As expected, periplocin or TRAIL treatment alone had little effects on the cleavage of BID, caspase 8, caspase 3, and PARP. The combination of periplocin and TRAIL treatments strongly increase the cleavage of all these apoptosis-related proteins (Figure
To further confirm the importance of caspase activation in periplocin-regulated cell apoptosis with or without TRAIL treatment, caspase inhibitors were added to HA22T/VGH cells prior to periplocin and/or TRAIL treatments. Inhibitors against caspase 3, caspase 8, and caspase 9 partially rescued cell survival repressed by periplocin and/or TRAIL treatments, and pan inhibitor against all three caspases completely blocked cell death induced by periplocin and/or TRAIL treatments (Figure
Another reported mechanism for sensitizing TRAIL-resistant cell lines to TRAIL treatment is through regulating proteins involved in apoptotic pathways [
Cotreatment of periplocin and TRAIL activated IAP. (a) The expression levels of Bax, Bad, Mcl-1, apaf-1, and caspase 9 in HA22T/VGH in response to 1
In addition to
Periplocin treatments affected tumor growth in xenograft
In Ki67 immunochemistry analysis, periplocin could inhibit the Ki67 expression in tumor samples (Figure
To further verify the periplocin-inhibited tumor growth
Periplocin is a cardiac glycoside structurally similar to digoxin. The pharmacological function of periplocin is also similar to digoxin and has been used to treat heart diseases. Nevertheless, digoxin was shown to block cancer growth through inhibiting HIF-1
TRAIL induces cell apoptosis via DR4 and DR5, activates Fas-associated death domain (FADD) and caspase 8, and signals through both mitochondria-dependent and -independent pathways [
The combination treatment of periplocin and TRAIL induced HCC cell apoptosis through activating IAP. IAPs are members of a protein family that regulate apoptosis. Currently there are 8 known members in this family, and X-linked inhibitor of apoptosis protein (XIAP) is the best characterized member in the family. XIAP blocks apoptosis by binding and inactivating caspase 3, 7, and 9 [
The expression of TRAIL by NK cells was demonstrated both
While purifying cortex periplocae, we identified six compounds with potential antitumor activity (Figure
In traditional herb medicine, patients are usually treated with multiple raw materials. Sometimes it is hard to purify one single active ingredient since multiple components are required to achieve therapeutic goals. In this study, we purified an active ingredient, periplocin, with cytotoxicity against HCC cells from cortex periplocae. Interestingly, maximum cytotoxicity against TRAIL-resistant HCC was achieved by combining the treatment of periplocin and TRAIL. The idea of the combination treatment is consistent with the concept of traditional medicine. Provided the mechanistic studies, the combination treatments of active ingredients from herb medicine and chemical synthesized compounds or protein drugs could be potential treatment options for drug-resistant cancers.
In this study, we demonstrated that periplocin could sensitize TRAIL-resistant HCC to TRAIL treatment, and the combination treatment of TRAIL and periplocin can induce apoptosis in TRAIL-resistant HCC. Furthermore, we showed that periplocin sensitized TRAIL-resistant HCC cell lines to TRAIL through the following two mechanisms. First, periplocin induced the expression of DR4 and FADD to activate proapoptotic signaling pathways. Second, the cotreatment of TRAIL and periplocin suppressed several IAP, which also led to the activation of proapoptotic signaling pathways. Our working model is shown in Figure
Mechanisms of resensitizing TRAIL-resistant HCC cells by periplocin. Periplocin can resensitize TRAIL resistant HCC by inducing the expression of DR4. Therefore, the combination treatments of TRAIL and periplocin can induce cell apoptosis through direct activation of caspase signaling and indirect inhibition of IAPs.
The authors claim no conflict of interests.