Background
Conventional cytotoxic anticancer drugs are commonly used in current standard cancer chemotherapies. However, the development of adverse effects is inherently difficult to avoid in their clinical use [
1,
2]. As they target nucleic acids or proteins involved in nucleic acid synthesis, DNA replication, transcription, and cell division, cytotoxic anticancer drugs inevitably damage normal proliferating cells besides cancer cells. In addition, drug resistance and tumor heterogeneity often limit the efficacy of monochemotherapies [
3]. More effective therapeutic strategies have been continuously pursued to overcome such limitations. Those include the combined use of multiple cytotoxic anticancer drugs or cytotoxic anticancer drugs with molecularly targeted drugs.
Pancreatic and biliary tract cancers are the most aggressive malignancies with poor prognoses [
4‐
6]. Due to the asymptomatic nature of the diseases at early stages, most patients are diagnosed at advanced stages that are not eligible for surgical resection. Gemcitabine (GEM), classified as an antimetabolite, is commonly used for drug treatments of these cancers [
4‐
6]. The first-line therapies for unresectable or metastatic diseases include GEM-based combined therapies, i.e., GEM with nab-paclitaxel or erlotinib for pancreatic cancer [
4,
5,
7,
8] and GEM with cisplatin or S-1 (tegafur, gimeracil, and oteracil potassium), or both, for biliary tract cancer [
6,
9,
10]. However, these current therapies often develop dose-limiting myelosuppression (such as leukopenia, neutropenia, and thrombopenia), achieving only modest life-prolonging effects [
4‐
6].
Cancer cells exhibit an increased uptake of amino acids as nutrients to satisfy their enhanced metabolic demands for rapid growth and proliferation. Furthermore, recent studies revealed the functional aspects of amino acids as signaling molecules. Especially, amino acids such as leucine are essential to activate mechanistic target of rapamycin complex 1 (mTORC1), a Ser/Thr-protein kinase complex that plays pivotal roles in regulating cell survival, growth, and proliferation and is often dysregulated in cancers [
11‐
13]. L-type amino acid transporter 1 (LAT1; SLC7A5) [
14], which preferentially transports large neutral amino acids, including most of the essential amino acids, is known to be upregulated in various types of cancers [
14,
15]. The high expression level of LAT1 is associated with the poor prognosis of patients with multiple cancer types, including pancreatic and biliary tract cancers [
16‐
18]. Due to its pathological function in cancer, LAT1 has been regarded as a rational target of molecularly targeted drugs.
Nanvuranlat (JPH203 or KYT-0353, abbreviated as NANV) is a LAT1-selective high-affinity inhibitor developed as the first-in-class anticancer agent [
19,
20]. The anticancer effects of NANV have been well-proven preclinically against cancer cells from various organs in vitro [
19,
21‐
30] and in vivo [
19,
24,
25,
27,
31‐
33]. Consistent with the predominant contribution of LAT1 in supplying cancer cells with essential amino acids, including leucine, treatment with NANV reduces mTORC1 activity in cancer cells [
21‐
24,
26,
27,
29‐
31,
33]. We have previously characterized the anticancer effects of NANV on pancreatic and biliary tract cancer cell lines [
29,
30]. Inhibition of LAT1 with NANV suppressed the uptake of all the eight primary substrates of LAT1 into cancer cells and inhibited the mTORC1 pathway, resulting in a global suppression of protein synthesis [
30]. Proteomics and phosphoproteomics revealed decreased phosphorylation of CDK1 and CDK2 [
29] by NANV as possible regulators involved in the cell cycle arrest at the G0/G1 phase caused by the inhibition of LAT1 [
25,
29,
33]. The first randomized phase II clinical trial of NANV monotherapy against pretreated, advanced, and refractory biliary tract cancers demonstrated a significant improvement in progression-free survival compared to placebo control (UMIN000034080) [
34]. Notably, the safety profile of NANV was confirmed to be comparable to that of a placebo without developing any severe adverse events that lead to discontinuation, dose reduction, or death.
Because NANV targets the cancer cell-specific molecule LAT1, its combinational use with cytotoxic anticancer drugs may enhance the treatment efficacy while mitigating the risk of leading adverse effects and resistance [
20]. We have previously shown that 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), a classical inhibitor of system L amino acid transporters including LAT1, in combination with the platinum drug cisplatin suppresses the growth of a head and neck squamous cell carcinoma cell line more strongly than by their single treatment [
35]. However, due to its limited affinity and selectivity to LAT1, BCH was not further developed as an anticancer drug. It is still open to question whether the new anticancer drug NANV exhibits enhanced anticancer activity in combination with cytotoxic anticancer drugs or not.
In the present study, we first tested the combinations of NANV with seven distinct types of cytotoxic anticancer drugs to inhibit the growth of pancreatic cancer MIA PaCa-2 cells. NANV showed significantly enhanced growth inhibitory effects with all the tested drugs, where a relatively strong enhancement of growth inhibition was obtained in combination with GEM. The combined effects were also verified in multiple pancreatic and biliary tract cancer cell lines. We performed analyses of apoptosis, cell cycle, and phosphorylation of amino acid-related signaling proteins to elucidate the pharmacological mechanisms underlying the combined effects. Finally, the significant combined effects of GEM and NANV were verified in cancer cell spheroid cultures. This study reveals the potential of LAT1 inhibitor NANV as a concomitant drug with GEM to treat malignant pancreatic and biliary tract cancers.
Methods
Anticancer drugs
5-Fluorouracil (5-FU, Wako), 7-ethyl-10-hydroxycamptothecin (an active metabolite of irinotecan) (SN-38, Selleck), paclitaxel (TXL, Wako), and nanvuranlat (NANV, J-Pharma Co., Ltd.) were dissolved in DMSO. Gemcitabine hydrochloride (GEM, Wako), oxaliplatin (L-OHP, Wako), and doxorubicin hydrochloride (DXR, Wako) were dissolved in water. For all the tested drug concentrations, a constant volume of 333-fold drug stock solutions was added to the medium. Cyclophosphamide monohydrate (CPA, Wako) was directly dissolved in the medium.
Cell culture
Pancreatic cancer HPAC (CRL-2119; ATCC), MIA PaCa-2 (JCRB0070; JCRB), PANC-1 (CRL-1469; ATCC), and SUIT-2 (JCRB1094; JCRB) cells and biliary tract cancer HuCCT1 (JCRB0425; JCRB), KKU-055 (JCRB1551; JCRB), KKU-100 (JCRB1568; JCRB), and KKU-213 (JCRB1557; JCRB) cells were cultured in RPMI-1640 supplemented with 10% FBS and 100 units/mL penicillin-100 µg/mL streptomycin. Cells were maintained in a humidified incubator at 37 °C supplied with 5% CO2.
Cell growth assay
Cells were seeded at 1.0 × 103 cells/well in 96-well plates (100 µL of medium/well). After 24 h of culture, the medium was replaced with a fresh medium containing the indicated concentrations of cytotoxic anticancer drug or NANV, or both. After 72 h of treatment, cell growth was measured by Cell Counting Kit-8 (Dojindo). Combined effects of drugs on cell growth were evaluated by the combination index (CI) based on the Bliss independence model using the following equation: CI = (EA+EB − EAEB)/EAB, where EA and EB represent the observed growth inhibition by drug A and B, respectively, and EAB by drug A combined with drug B. When CI is under, above, or equal to 1, the combined effects was judged as synergistic, antagonistic, or additive, respectively.
Apoptosis assay
Cells were seeded at 3.0 × 104 cells/well in 6 well plates (3 mL of medium/well). After 24 h, the medium was replaced with a fresh medium containing GEM or NANV, or both. After 72 h of incubation, apoptosis was analyzed by Muse™ Cell Analyzer (Millipore) using Muse™ Annexin V and Dead Cell kit. Annexin V and Dead Cell kit. Apoptotic rate (%) was expressed as the sum of the percentages of early (Annexin V-positive/7-AAD-negative) and late (Annexin V-positive/7-AAD-positive) apoptotic cells.
Cell cycle analysis
Cells were seeded at 4.5 × 105 cells/dish in 100 mm dishes containing 15 mL of medium and cultured for 48 h. Then, the cells were incubated for 24 h with a fresh medium containing GEM or NANV, or both. Cell cycle analysis was performed by Muse™ Cell Analyzer (Millipore) using Muse™ Cell Cycle kit.
Western blot
Cells were seeded at 4.5 × 10
5 cells/dish in 100 mm dishes containing 15 mL of medium and cultured for 48 h. Then, the cells were incubated for 24 h with a fresh medium containing GEM or NANV, or both. Western blot was performed as described previously [
30]. Primary antibodies used are as follows: anti-β-actin (66009-1-Ig) from Proteintech; anti-phospho-Ser240/244-S6 ribosomal protein (5364), anti-S6 ribosomal protein (2217), anti-phospho-Ser51-eIF2α (3398), anti-eIF2α (5324), anti-phospho-Thr37/46-4EBP1 (2855), and anti-4EBP1 (9452) from Cell Signaling Technology.
Spheroid culture
Cells were seeded in 96-well clear round bottom ultra-low attachment microplates (Corning, 7007) at 1.0 × 103 cells in 100 µL/well of the medium. After centrifugation at 300×g for 10 min at 25 °C to sediment the cells, 100 µL of medium containing 10% (v/v) Matrigel (Falcon, 354230) was added to each well. Then the cells were cultured in a humidified incubator at 37 °C supplied with 5% CO2 to induce spheroid formation. After incubation for 72 h, 100 µL of the medium was replaced by 100 µL of a fresh medium containing either GEM or NANV, or both, at twice the final concentration (Day 0). On Day 3 and 5, 100 µL of the medium was replaced by 100 µL of a fresh medium containing the drug(s) at the indicated final concentrations. Bright-field images of spheroids were taken every 24 h by microscope (Leica, DMi1, MC120 HD). The projected area of spheroids was calculated using ImageJ software (NIH).
Data reproducibility and statistical analysis
All the experiments were repeated at least twice to ensure the reproducibility of the results. Statistical analyses were performed with GraphPad Prism9 (GraphPad software). Differences were considered significant when p-values were < 0.05. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant.
Discussion
In this study, we first examined the combinations of seven distinct types of cytotoxic anticancer drugs with an amino acid transporter LAT1 inhibitor, nanvuranlat (NANV; JPH203 or KYT-0353), on the growth of pancreatic cancer MIA PaCa-2 cells (Fig.
1B). All the tested combinations showed significantly enhanced growth inhibitory effects compared to their single treatments. The combined effects were suggested to be primarily additive under the current experimental conditions. Whereas we previously reported the combination effects of BCH and cisplatin against head and neck cancer cells [
35], BCH is a compound with a broad specificity over system L amino acid transporters [
39]. The obtained anticancer effects thus cannot be specifically attributed to the inhibition of LAT1. For the first time, this study revealed the combined growth inhibitory effects specifically obtained by LAT1 inhibition with various types of cytotoxic anticancer drugs. Considering that NANV has demonstrated anticancer effects against cancer cells derived from various organs in preclinical studies [
19,
21‐
30], these results suggest the potential of NANV for broad clinical applications against multiple types of cancers in combination with cytotoxic anticancer drugs. Among the tested drugs, NANV exhibited relatively high combined effects with GEM, CPA, SN-38, and DXR. Therefore, we selected combining GEM with NANV for further evaluation against pancreatic and biliary tract cancer cells because GEM-based drug therapies are standard treatments for these refractory cancer types but remain ineffective [
4‐
6]. The favorable outcomes of the first phase II clinical trial of NANV monotherapy in pretreated, advanced, and refractory biliary tract cancers encouraged us to pursue this possibility (UMIN000034080) [
34]. As a result, significant enhancement of the growth inhibitory effects by combining GEM and NANV was demonstrated in all the tested pancreatic and biliary tract cancer cell lines (four cell lines for each cancer type) (Figs.
1B and
2). The combination effects were observed not only in two-dimensional cultures but also in spheroid cultures of cancer cells (Fig.
6).
To elucidate the molecular basis for the combined effects of GEM and NANV, we performed analyses of the cell cycle, apoptosis, and amino acid-related signaling. The obtained overall results revealed no apparent enhancement in the pharmacological activities of each drug under the current experimental conditions. Consistent with the previous reports [
38], the single treatment with GEM induced cell cycle arrest at the S phase and apoptosis. NANV alone induced cell cycle arrest at the G0/G1 phase and did not induce apoptosis, as shown in previous research [
25,
29,
33]. The combination of GEM and NANV caused the cell cycle arrest at the S phase and induced apoptosis to similar levels as GEM alone in MIA PaCa-2, SUIT-2, and KKU-100 cells (Figs.
3 and
4). Therefore, GEM is supposed to influence the cell cycle and apoptosis more dominantly than NANV in their combination. An exceptional observation was made in the cell cycle analysis of the KKU-055 cell. The proportion of cells at the S phase was increased by combining GEM with NANV, but not by GEM alone. Notably, the G0/G1 cells were at a similar level as the untreated control under the combined treatment, whereas the G2/M cells were significantly decreased in KKU-055 cells. Although the details remain to be elucidated, these observations suggest that the increase of S phase cells in KKU-055 cells by the combined treatment with GEM and NANV cannot be simply interpreted as the enhanced activity of GEM that induces the S phase arrest by decreasing cells at the G0/G1 phase. Consistently, NANV did not potentiate the apoptosis-inducing activity of GEM in KKU-055 cells. Treatments with NANV altered the phosphorylation levels of proteins in amino acid-related signaling pathways to similar levels, irrespectively to the presence or the absence of GEM (Fig.
5). The identified changes in the phosphorylation in mTORC1 and GAAC pathways suggest the suppression of protein synthesis, representing the pharmacological activity of NANV without noticeable augmentation by GEM. These results indicate that GEM and NANV mostly independently exert their anticancer activities even in combination.
This study investigated the combination of GEM and NANV at a single dose set. We focused on revealing the general molecular mechanisms underlying the combination effects using multiple pancreatic and biliary tract cell lines. Conversely, the concentrations and ratio of the two drugs remain to be optimized to attain the best combination effects. Furthermore, we adopted the Bliss independence model [
36] to evaluate the drug combination effects because the mechanisms of action of cytostatic NANV and cytotoxic anticancer drugs are regarded as primarily independent. However, all the available reference models still present some limitations and do not perfectly fit the actual experimental conditions [
37,
40]. The analyses of GEM and NANV in this study implied that their detailed pharmacological activities are not completely independent and partially interfere with each other, as exemplified in their effects on the cell cycle, where the effects of NANV to induce the G0/G1 arrest was generally masked when combined with GEM (Fig.
4). The cooperative use of multiple theoretical and experimental methods [
37,
40] thus would be important to reinforce the significance of our findings in future studies.
Nevertheless, because NANV is the first-in-class anticancer drug targeting LAT1, the discovery of GEM as a preferable combination partner holds significant implications for its future clinical development. The findings of this study may contribute to developing novel therapeutic strategies with GEM, which is currently widely used for pancreatic and bile duct cancers. Notably, cancer cell-specific cytostatic anticancer activities of NANV may pave the way to circumvent the problems of adverse effects and drug resistance posed by GEM (and other cytotoxic anticancer drugs). Significant combination effects of a mTORC1 inhibitor, temsirolimus, and GEM have been reported previously in an animal model of pancreatic cancer [
41], while failed to show clinical efficacy in the first phase I/II study [
42]. It has also been reported that another mTORC1 inhibitor, everolimus, shows more pronounced antiproliferative effects against GEM-resistant pancreatic cancer cells than against GEM-sensitive pancreatic cancer cells [
43] and exhibits synergistic antiproliferative effects with GEM against biliary tract cancer cells [
44]. In addition to inhibiting mTORC1 by blocking the essential input of amino acid signals, NANV induces the depletion of amino acids as biosynthetic materials and suppresses the global translation in cancer cells [
30]. Thus, combining NANV with GEM may exhibit robust and multifaceted anticancer effects based on such broad pharmacological activities. A particularly tempting speculation in this regard would be that NANV, co-administrated with GEM, inhibits cancer cell growth by generally suppressing protein synthesis and prevents the acquisition of drug resistance by abolishing the expression of proteins involved in the resistance to GEM [
38,
45]. Future studies should also investigate such possible mechanistic convergence in their anticancer activities that may lead to better combination effects.
Conclusions
This study provides the primary evidence for the combinational effects of gemcitabine with a novel molecularly targeted drug, nanvuranlat, that may propose effective treatments for malignant pancreatic and bile duct cancers. The two drugs, when combined, additively suppressed the growth of cancer cells by exhibiting their pharmacological activities largely independently under the tested conditions. To further explore the in vivo relevance of our findings, detailed conditions for drug treatments, especially the concentrations and ratio of the two drugs, need to be further optimized to accomplish the best combination effects. Validation of the combination effects based on the two or more mutually compensative evaluation methods will also be particularly important. Results of such future studies will provide valuable information to extrapolate and enhance the combined effects of gemcitabine and nanvuranlat in in vivo animal models and clinical settings.
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