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Cancer stem cells (CSCs) are a small proportion of tumor cells that may be responsible for tumor metastasis and recurrence. Our recent research indicated that longikaurin A (LK-A) exhibited anti-tumor activity in nasopharyngeal carcinoma (NPC) both
Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in South China (
The CSC hypothesis proposes that a small subset of cancer cells has the properties of self-renewal, differentiation and resistance to chemotherapy or radiotherapy (
Several studies have revealed that nigericin (
We used a previously described method to obtain LK-A from the leaves of
S18 and S26 cells are clones of the human NPC cell line CNE2. A stable radioresistant NPC cell line (Sune2-IR) and its parental cell line (Sune2), and the 5–8F NPC cell line, were supplied by and maintained in the State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center (Guangzhou, China) in RPMI 1640 medium supplemented with 5% fetal bovine serum (FBS) (Invitrogen; Thermo Fisher Scientific, Inc.), penicillin (100 U/ml) and streptomycin (100 U/ml). The NPC cell lines were incubated at 37°C in 5% CO2/95% air.
S18 and S26 cells were plated in triplicate at a density of 200 cells per well in ultra-low attachment 6-well plates (Corning Incorporated, Corning, NY, USA), and then cultured in DMEM/F12 with 20 ng/ml recombinant human bFGF, 20 ng/ml recombinant human EGF and B-27® Supplement. The spheres were collected after 7 days and counted under a light microscope. Then, we dissociated the primary sphere cells into single-cell suspensions, which were cultured to allow the regeneration of spheres.
SP cell analysis and isolation were performed by fluorescence-activated cell sorting (FACS) (Beckman Coulter, Inc., Brea, CA, USA). Before SP cell analysis, cells were pretreated with different concentrations of LK-A for 48 h. Subsequently, the cells were resuspended at a density of 1×106 cells/ml in RPMI 1640 supplemented with 2% FBS. Then, cells were incubated with 5 µg/ml Hoechst 33342 (Sigma-Aldrich; Merck Millipore) either alone or with 100 µg/ml verapamil (Sigma-Aldrich; Merck Millipore) at 37°C in the dark for 90 min. Cells were washed, centrifuged at 1811 ×
First, 2,000 cells were seeded into 96-well plates, incubated overnight and then treated with various concentrations of LK-A for 48 h. Then, 20 µl of MTT (5 mg/ml) was added to each well, and the plate was incubated at 37°C for 4 h. Subsequently, the supernatant was carefully removed, and 150 µl/well DMSO was added to dissolve the formazan crystals. The absorbance of the soluble product was measured with a microplate spectrophotometer at 490 nm (µQuant™; Biotek Instruments, Inc., Winooski, VT, USA). This experiment was performed in six replicates and repeated three times. We calculated the percentage of cell viability for each concentration of LK-A by using the following formula: Cell viability (%) = A570 nm (sample) / A570 (control DMSO) × 100. The half maximal inhibitory concentration (IC50) was determined with GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA).
S18 and S26 cells were counted, plated in triplicate at 2,000 cells per well (200 ml) in 96-well plates, and allowed to grow overnight. For the experiment group, a concentration gradient of cisplatin (DDP; Jiangsu Hansoh Pharmaceutical Group Co., Ltd., Lianyungang, China) was added to the wells. For the combination groups, 1.0 µM LK-A was mixed with a concentration gradient of DDP and then added to the wells. Cell viability was measured 48 h later by adding an MTT solution. The observation value was detected at 490 nm. The results from the assays were analyzed for the combination effect between LK-A and DDP according to the method described by Jin (
The NPC cells were seeded into six-well plates with 400 cells per well. Twenty-four hours after plating, LK-A was added at a concentration of 0.4 µM. After 24 -h exposure to LK-A, the Sune2 cells and the radioresistant Sune2-IR cells were irradiated at doses of 0, 1, 2 and 3 Gy. Immediately following irradiation, the cells were incubated for 7 days at 37°C to allow colony formation in a 5% CO2 environment. After 10 days, the colonies were washed with PBS, fixed with pure ethanol and stained with 0.5% crystal violet. Colonies containing >50 cells were counted as clonogenic survivors. Each point on the survival curves represents the mean surviving fraction (SF) from at least three dishes. The equation SF=1-(1-e-D⁄D0)N was applied to calculate several parameters, including the cellular radiosensitivity (mean lethal dose, D0), the capacity for sublethal damage repair (quasi-threshold dose, Dq) and the extrapolation number (N). Those parameters were used to calculate the sensitization enhancement ratio (SER) and the SF2 (the SF after irradiation at a dose of 2 Gy).
Cells were seeded into 6-well plates and incubated overnight. Then, the cells were treated with various concentrations of LK-A for 48 h. We used a previously described western blotting method (
Data are presented as the mean ± standard deviation. GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA) was used to determine IC50, and SigmaPlot v10.0 (Systat Software, Inc., San Jose, CA, USA) was used to construct cell survival curves. Student's t-test was used to compare the means of various groups. P<0.05 was considered to indicate a statistically significant difference.
To investigate the effect of LK-A on the self-renewal ability of NPC CSCs, we evaluated the sphere-forming capacity after adding LK-A to S18 and S26 cells growing in serum-free non-adherent medium. The result revealed that interference by LK-A resulted in markedly diminished sphere size and number of spheres in a dose-dependent manner in both S18 and S26 cells (
To further investigate the effect of LK-A on second-generation spheres, after spheres formation, we dissociated the sphere cells into single cells, and we cultivated these single cells in serum-free non-adherent culture with various concentrations of LK-A again. The first-generation spheres were capable of generating second-generation spheres, suggesting that NPC sphere-generated cells have the capacity of self-renewal (
SP cells are one subpopulation of the cancer cells that effluxes the DNA binding dye Hoechst 33342 out of the cell membrane. Previous studies have shown that the SP cells have stem cell characteristics and enrich the stem cell population in NPC (
CSCs are believed to be the cause of chemotherapy resistance (
Despite improvements in radiation technology, local recurrence or metastasis occurs in a high proportion of NPC patients due to radioresistance (
It was reported that silencing fibronectin extra domain A (EDA) enhances radiosensitivity in NPC (
As it is well known that there are not reliable CSC markers in NPC cells (
As aforementioned, LK-A may sensitize Sune2 cells to radiation and reversed the radioresistance of Sune2-IR cells through influencing fibronectin protein expression. Fibronectin is known to play important roles in angiogenesis, lymphangiogenesis and metastasis in malignant tumors (
Currently, the main treatments of NPC are radiotherapy and platinum-based chemotherapy (
As a natural ent-kaurene diterpenoid, LK-A has been reported to induce apoptosis in multiple myeloma H929 cells, hepatocellular carcinoma cells and NPC cells (
In addition to serving as a potential therapeutic for NPC CSCs, our results suggest that LK-A exerts additive effects with DDP on NPC cells (
Previous studies indicated that silencing fibronectin EDA increases radiosensitivity in NPC involving the focal adhesion kinase/Akt/c-Jun N-terminal kinase pathway (
Previous research about LK-A indicated that LK-A induces G2/M phase arrest via downregulation of S-phase kinase-associated protein 2 (Skp2) and apoptosis induction in hepatocellular carcinoma cells (
Furthermore, we explored the effects of LK-A on stem cell markers. Despite the fact that LK-A treatment decreased the percentage of SP in NPC cells, the expression of ABCG2 protein was not affected (data not show). However, we found that LK-A reduced the expression of c-myc in a dose-dependent manner (
In summary, LK-A could target CSCs in NPC cells, and it could enhance the efficacy of chemotherapy drugs and radiotherapy
(A and B) LK-A reduced the number and size of the primary spheroids formed. Magnification, ×200. S18 and S26 cells were plated in single-cell suspensions at a density of 100 cells/ml in cancer stem cell medium (Dulbecco's modified Eagle's medium/F12, human epidermal growth factor and B27 supplement) and treated with various doses of LK-A for 7 days. (C and D) Primary spheres were harvested, and then re-plated after dilution to analyze the subsphere forming ability. Magnification, ×200. LK-A, longikaurin A.
LK-A decreases the percentage of SP cells in the S18 cell line. (A and B) Percentages of SP cells in the (A) S18 and (B) S26 cell lines after treatment with LK-A. The SP cell profiles in the presence of verapamil are shown in the bottom panels. The percentages of SP cells are indicated. LK-A, longikaurin A; SP, side population.
Cytotoxic effect of cisplatin alone or in combination with LK-A. (A-E) Shifting of dose-response curves of cisplatin by LK-A. Three nasopharyngeal carcinoma cell lines, (A) S18, (B) S26 and (C) 5–8F, were assayed by MTT assay. (D) LK-A potentiated the effect of cisplatin in the three cell lines. Q=Ea+b/(Ea+Eb-EaxEb), where Ea+b, Ea and Eb are the average effects of the combination treatment, LK-A only and cisplatin only, respectively. Q<0.85 indicates antagonism, 0.85≤Q<1.15 indicates additivity and Q≥1.15 indicates synergism. LK-A, longikaurin A; DDP, cisplatin.
LK-A pretreatment sensitized Sune2 cells to radiation and reversed the radioresistance of Sune2-IR cells. (A) LK-A was added in concentrations of 0.4 µmol/l. After 24 h exposure to LK-A, the Sune2 cells and the radioresistant Sune2-IR cells were irradiated at doses of 0, 1, 2 and 3 Gy. After 10 days, the colonies were fixed with pure ethanol and stained with 0.5% crystal violet. (B) Clonogenic survival assays of Sune2 and Sune2-IR cells pretreated with LK-A. Surviving fractions were calculated by the number of colonies divided by the number of seeded cells multiplied by the plating efficiency. (C) SER was calculated from the data shown in A. LK-A, longikaurin A; SER, sensitization enhancement ratio.
Longikaurin A pretreatment downregulated the protein expression of the (A) c-myc and (B) fibronectin in the S18 and S26 cell lines. (C) Expression of fibronectin in the Sune2 and Sune2-IR cell lines. Numbers in the figure refer to mean grey value.
IC50 value for DDP in the presence or absence of LK-A in S26, S18 and 5–8F cells.
IC50 value for DDP in the cell lines (48 h) | S18 | S26 | 5-8F |
---|---|---|---|
Control | 10.10±1.00 µM | 11.45±1.06 µM | 8.00±0.90 µM |
1 µM LK-A | 7.00± 0.85 µM | 7.12±0.85 µM | 2.65±0.42 µM |
IC50, half maximal inhibitory concentration; LK-A, longikaurin A; DDP, cisplatin.
Clonogenic survival parameters of Sune2 and Sune2-IR cells after LK-A exposure.
Cell lines | Groups | D0 | Dq | SF2 | N | SER (Dq) |
---|---|---|---|---|---|---|
Sune2 | Control | 2.58 | 1.17 | 0.59 | 1.25 | 1.16 |
0.4 µM LK-A | 2.48 | 1.01 | 0.53 | 1.10 | ||
Sune2-IR | Control | 2.22 | 1.51 | 0.63 | 2.04 | 1.14 |
0.4 µM LK-A | 2.15 | 1.33 | 0.58 | 1.76 |
LK-A, longikaurin A; D0, mean lethal dose; Dq, quasi-threshold dose; SF, surviving fraction; SF2, SF after irradiation at a dose of 2 Gy; N, extrapolation number; SER, sensitization enhancement ratio.