Combination effects of amino acid transporter LAT1 inhibitor nanvuranlat and cytotoxic anticancer drug gemcitabine on pancreatic and biliary tract cancer cells

Abstract

Background: Cytotoxic anticancer drugs widely used in cancer chemotherapy have some limitations, such as the development of side effects and drug resistance. Furthermore, monotherapy is often less effective against heterogeneous cancer tissues. Combination therapies of cytotoxic anticancer drugs with molecularly targeted drugs have been pursued to solve such fundamental problems. Nanvuranlat (JPH203 or KYT-0353), an inhibitor for L-type amino acid transporter 1 (LAT1; SLC7A5), has novel mechanisms of action to suppress the cancer cell proliferation and tumor growth by inhibiting the transport of large neutral amino acids into cancer cells. This study investigated the potential of the combined use of nanvuranlat and cytotoxic anticancer drugs.

Methods: The combination effects of cytotoxic anticancer drugs and nanvuranlat on cell growth were examined by a water-soluble tetrazolium salt assay in two-dimensional cultures of pancreatic and biliary tract cancer cell lines. To elucidate the pharmacological mechanisms underlying the combination of gemcitabine and nanvuranlat, we investigated apoptotic cell death and cell cycle by flow cytometry. The phosphorylation levels of amino acid-related signaling pathways were analyzed by Western blot. Furthermore, growth inhibition was examined in cancer cell spheroids.

Results: All the tested seven types of cytotoxic anticancer drugs combined with nanvuranlat significantly inhibited the cell growth of pancreatic cancer MIA PaCa-2 cells compared to their single treatment. Among them, the combined effects of gemcitabine and nanvuranlat were relatively high and confirmed in multiple pancreatic and biliary tract cell lines in two-dimensional cultures. The growth inhibitory effects were suggested to be additive but not synergistic under the tested conditions. Gemcitabine generally induced cell cycle arrest at the S phase and apoptotic cell death, while nanvuranlat induced cell cycle arrest at the G0/G1 phase and affected amino acid-related mTORC1 and GAAC signaling pathways. In combination, each anticancer drug basically exerted its own pharmacological activities, although gemcitabine more strongly influenced the cell cycle than nanvuranlat. The combination effects of growth inhibition were also verified in cancer cell spheroids.

Conclusions: Our study demonstrates the potential of first-in-class LAT1 inhibitor nanvuranlat as a concomitant drug with cytotoxic anticancer drugs, especially gemcitabine, on pancreatic and biliary tract cancers.

Keywords: Amino acid transporter; Cancer chemotherapy; Combination therapy; Cytotoxic anticancer drugs; Essential amino acids; Gemcitabine; LAT1; Large neutral amino acids; Molecularly targeted drugs; SLC7A5.

Fig. 1

Inhibition of cell growth by single or combined treatment with cytotoxic anticancer drugs and nanvuranlat. MIA PaCa-2 cells were treated with drugs for 72 h. Cell growth was measured by Cell Counting Kit-8 (WST-8). Data were normalized by untreated controls and shown as mean ± SD (n = 8, technical replicates in a single experiment). A Concentration-dependent cell growth inhibition by single treatment with seven cytotoxic anticancer drugs and nanvuranlat (NANV). B Growth of MIA PaCa-2 cells treated with cytotoxic anticancer drugs or NANV, or both. Drugs were used at following concentrations: 5-FU, 3.5 µmol/L; GEM, 13 nmol/L; L-OHP, 2 µmol/L; CPA, 2.5 mmol/L; SN-38. 2.7 µmol/L; DXR, 20 nmol/L; TXL, 2.3 µmol/L; and NANV, 3 µmol/L. Statistical significance was evaluated by one-way ANOVA followed by Tukey's post-test. Combination indices (CI) were calculated based on the Bills independence model

Fig. 2

Inhibition of cell growth by single or combined treatment with gemcitabine and nanvuranlat in multiple pancreatic and biliary tract cancer cell lines. Pancreatic cancer (HPAC, PANC-1, and SUIT-2) and biliary tract cancer (HuCCT1, KKU-055, KKU-100, and KKU-213) cells were treated with GEM or NANV, or both, for 72 h. Cell growth was measured by Cell Counting Kit-8 (WST-8). Data were normalized by untreated controls and shown as mean ± SD (n = 8, technical replicates in a single experiment). Cells were treated with drugs at following concentrations: HPAC cells (GEM, 10 nmol/L; NANV, 6 µmol/L), PANC-1 cells (GEM, 150 nmol/L; NANV, 30 µmol/L), SUIT-2 cells (GEM, 3 nmol/L; NANV, 12 µmol/L), HuCCT1 cells (GEM, 20 nmol/L; NANV, 1.3 µmol/L), KKU-055 cells (GEM, 10 nmol/L; NANV, 0.9 µmol/L), KKU-100 cells (GEM, 6.5 nmol/L; NANV, 8 µmol/L), and KKU-213 cells (GEM, 40 nmol/L; NANV, 7 µmol/L). Statistical significance was evaluated by one-way ANOVA followed by Tukey's post-test. Combination indices (CI) were calculated based on the Bills independence model

Fig. 3

Induction of apoptosis by single or combined treatment with gemcitabine and nanvuranlat. Apoptosis induction was measured in MIA PaCa-2, SUIT-2, KKU-055, and KKU-100 cells treated with GEM or NANV, or both, for 72 h. Cells were treated with drugs at the following concentrations: MIA PaCa-2 cells (GEM, 13 nmol/L; NANV, 3 µmol/L), SUIT-2 cells (GEM, 3 nmol/L; NANV, 12 µmol/L), KKU-055 cells (GEM, 10 nmol/L; NANV, 0.9 µmol/L), and KKU-100 cells (GEM, 6.5 nmol/L; NANV, 8 µmol/L). Statistical significance was evaluated by one-way ANOVA followed by Tukey's post-test. Data were shown as mean ± SD (n = 3, biological replicates)

Fig. 4

Effects of single or combined treatment with gemcitabine and nanvuranlat on cell cycle. MIA PaCa-2, SUIT-2, KKU-055, and KKU-100 cells were treated with GEM or NANV, or both, for 24 h and subjected to the cell cycle analysis. Cells were treated with drugs at the following concentrations: MIA PaCa-2 cells (GEM, 13 nmol/L; NANV, 3 µmol/L), SUIT-2 cells (GEM, 3 nmol/L; NANV, 12 µmol/L), KKU-055 cells (GEM, 10 nmol/L; NANV, 0.9 µmol/L), and KKU-100 cells (GEM, 6.5 nmol/L; NANV, 8 µmol/L). Data were shown as mean ± SD (n = 3, biological replicates). Statistical significance was evaluated by two-way ANOVA followed by Tukey's post-test

Fig. 5

Effects of single or combined treatment with gemcitabine and nanvuranlat on amino acid signaling. MIA PaCa-2, SUIT-2, KKU-055, and KKU-100 cells were treated with GEM or NANV, or both, for 24 h, and analyzed by Western blot. Phosphorylated and total proteins of S6 ribosomal protein, 4EBP1, and eIF2α were detected. Cells were treated with drugs at the following concentrations: MIA PaCa-2 cells (GEM, 13 nmol/L; NANV, 3 µmol/L), SUIT-2 cells (GEM, 3 nmol/L; NANV 12 µmol/L), KKU-055 cells (GEM, 10 nmol/L; NANV 0.9 µmol/L), KKU-100 cells (GEM, 6.5 nmol/L; NANV, 8 µmol/L)

Fig. 6

Inhibition of spheroid growth by single or combined treatment with gemcitabine and nanvuranlat. Spheroids of MIA PaCa-2 and KKU-055 cells were treated with GEM or NANV, or both, at the following concentrations: MIA PaCa-2 cells (GEM, 15 nmol/L; NANV, 30 µmol/L) and KKU-055 cells (GEM, 12.5 nmol/L, NANV, 1 µmol/L). Drug treatment was started on Day 0. The half-volume medium exchange was done on Day 3 and Day 5. A Representative bright-field images of spheroids on Day 0 and Day 7. Scale bar: 500 μm. B Quantification of the spheroid growth. The projected area size of each spheroid on each day was calculated from the bright-field image and normalized by Day 0. Data were shown as mean ± SD (n = 10, technical replicates in a single experiment). Statistical significance was evaluated by two-way ANOVA followed by Tukey's post-test