非小細胞肺がん細胞株におけるチューブリン重合とアセチル化を介したゲムシタビンとパクリタキセルの相乗効果

概要

Lung cancer is a highly lethal form of malignancy. Approximately 85%-90% of lung cancer cases were non-small cell lung cancer (NSCLC). Although the treatment of lung cancer has provided novel molecular-targeted therapies and immunotherapies, anti-cancer cytotoxic drugs are still relevant.

Gemcitabine (GEM) works as a single or combination chemotherapy by inhibiting DNA synthesis. Paclitaxel (PTX) is a microtubule-interfering drug that promotes tubulin polymerization. Combining those drugs was essential in the treatment of advanced NSCLC because of their different action mechanisms and partially non-overlapping toxicities. Besides, Nanoparticle albumin-bound-paclitaxel (NP) is a novel drug that delivers PTX optimally based on the pharmacokinetic profile.

Previous studies showed that PTX reinforces GEM action by increasing deoxycytidine kinase (dCK), while NP reduces cytidine deaminase (CDA). This study aimed to elucidate a potential effect of GEM combined with PTX and NP on the proliferation of two NSCLC cell lines, A549 and H520.

Our study demonstrated that A549 was the most sensitive to anti-cancer drugs. The half- maximal inhibitory concentration (IC50) of GEM in A549 and H520 was 6.6 nM and 46.1 nM, respectively, while the PTX`s IC50 in A549 and H520 was 1.35 nM and 7.59 nM, respectively.

The standard in determining synergism or not is using the combination index (CI). The range of CI <0.1, 0.1-0.3, 0.3-0.7, 0.7-0.85, 0,85-0.9, 0.9-1.1, 1.1-1.2, 1.2-1.45, 1.45-3.3, and>10 describes potent synergism, strong synergism, synergism, moderate synergism, slight synergism, nearly additive, mild antagonism, moderate antagonism, antagonism, strong antagonism, and extreme antagonism, respectively. The mean CI for every fraction affected (Fa, % inhibition of cellular proliferation) at 0.5, 0.75, and 0.9 is the final CI.

Among the sequences explored (GEM → PTX, PTX → GEM, and GEM and PTX simultaneously (GEM+PTX)), GEM→PTX sequences produced a mean CI < 1 in both cell lines. The strongest synergism was apparent in H520 cells with a CI of 0.10741 at 0.5 Fa. This finding is of considerable clinical importance even though synergy at a high-level Fa for cancer treatment is more critical.

To measure the acetylated tubulin protein, we use western blot analysis and immunofluorescent staining for tubulin polymerization. The GEM→PTX sequence produced the most expression of acetylated tubulin protein and enhanced α-tubulin polymerization. This particular sequence also worked synergistically to suppress tumor growth in vivo.

How can we predict the synergism and antagonism of this combination? An earlier pharmacodynamic study showed that a low dose of GEM (1 ng/ml) induced cell cycle arrest in phase S of the cell cycle, and assisted the efficacy of the subsequent drug. Also, a low dose of PTX can stabilize microtubule during mitosis at phase G2-M and lead to apoptosis rather than necrosis. The other studies described that low PTX might arrest the cell only at phase G1 and also both G2-M and G1.

Furthermore, low PTX may induce aberrant mitosis resulting in aneuploidy and lead to apoptosis. We assume that the administration of small GEM as the first drug in combination produces a transient accumulation cell in phase S. It will facilitate the efficacy of the next pill, a low dose of PTX. In reverse sequence, exposure to low PTX probably caused cells to arrest in phase G1, thereby the number of cells in phase S was reduced, leading to the insufficiency of cell cycle-specific cytotoxicity of GEM.

Administration low GEM as a first drug also generates synergism that represented by apoptosis and migration assay. Apoptosis was more pronounced in the sequence combination in which GEM preceded GEM+PTX. The migration rate of cells was also decreased after a scratch-made and followed for 48 hours. The increases in apoptotic cell and deceleration of migration assay may be related to the scheduling of drug combinations.

Growth inhibition in GEM→NP seemed more favorable. The maximum tolerated dose (MTD), and the lethal dose of NP was 120-240 mg/kg and 240mg/kg, respectively. More cells arrested by GEM in phase S will facilitate the low treatment of NP (50mg/kg) to induce cell apoptosis and suppress tumor growth. GEM works effectively in phase S so that the use of NP as the first drug or concurrent combination will decrease the activities of GEM. Hence, the single purpose of NP might inhibit the tumor growth similar to group NP → GEM and simultaneous sequence.

Microtubule stabilization, resulting in cell apoptosis, is a cornerstone that underlies drug-drug interaction. Microtubules, which consist of α and β tubulin, are highly dynamic structures representing the target of tubulin-binding anti-cancer drugs. PTX interacts with an amino-terminal region of tubulins against depolymerization. Even at low concentrations, PTX can still exert the ability to suppress the dynamics of a microtubule. Tubulin acetylation is an indicator of microtubule stability. In contrast, histone deacetylase 6 (HDAC6), a class IIb deacetylase, is known to deacetylate substrates such as tubulin.

A previous study combining trichostatin A and PTX produces a significant increase in tubulin acetylation and microtubule stabilization. On the other hand, GEM can influence microtubules indirectly. For example, GEM can play a vital role in apoptosis driven by caspase-3. The cleavage of the C-terminal ubiquitin-binding zinc-finger of HDAC6 will promote apoptosis. Therefore, GEM reinforcing the anti-cancer effect of PTX by acetylation and polymerization of microtubules.

In conclusion, the present study provides evidence that the synergistic drug combination of low GEM and low PTX/NP might inhibit the growth of NSCLC cell lines. Furthermore, the administration of small GEM as a first drug in the combination was proven to enhance the anti- tumor activity of low PTX by modulating tubulin acetylation and microtubule polymerization. The GEM→PTX/NP sequence may represent a promising candidate regimen for the treatment of advanced NSCLC.