Cancer is a major health problem and one of them is metastasis. Metastatic cancer causes
> 400,000 deaths annually, and these deaths are expected to be more than double by 2040. Cancer pain is
a major problem associated with the clinical management of cancer. Some analgesic targets, such as ion
channels and receptors, are also involved in the cancer process, so the use of some of these analgesics
can have an impact on cancer metastasis. Gabapentin as an adjuvant analgesic is used as the fi rst line of
treatment for neuropathic pain in cancer pain. The purpose of this study was to determine the potential
role of gabapentin in modulating cellular apoptosis for protecting the progression and metastatic processes
in cancer cell culture.

The research design used was an in vitro experimental randomized design. There were four
groups in this study, namely three treatment groups and one control group with three repetitions per
group. Gabapentin was given to each treatment group with graded doses of 1,000 ng/mL, 2,000 ng/mL,
and 4,000 ng/mL, then the apoptotic index was tested. The mean apoptotic index was analyzed by analysis
of variance (ANOVA) test and followed by the least signifi cant differences level (α) of 5% (0.05).

The ANOVA test showed that there was a signifi cant difference in the percentage of the apoptotic
index test in the four groups based on the fl ow cytometry method, with a P value < 0.05.

The study concludes that giving gabapentin is effective in increasing the apoptotic index of
cancer cell cultures.

apoptosis index, cancer cell culture, gabapentin, metastatic process

Cancer is the leading cause of death in the world, and it is estimated that cancer occurs in 8 million people in 2012 worldwide with 14 million new cases diagnosed each year [1]. Based on data from the 2017 National Cancer Institute, in 2016 an estimated 1,685,210 cases of cancer were diagnosed in the United States, and 595,690 people died from the disease. The most common cancers in 2016 were breast cancer, lung and bronchial cancer, prostate cancer (PCA),colon and rectal cancer, bladder cancer, skin melanoma, non-Hodgkin lymphoma, thyroid cancer, kidney cancer, leukemia, endometrial cancer, and pancreatic cancer. The number of new cases of cancer (cancer incidence) is 454.8 per 100,000 men and women per year (based on 2008–2012 cases) [2].

According to the 2013 Basic Health Research, nationally the prevalence of cancer in the population of all ages in Indonesia in 2013 was 1.4% or an estimated 347,792 people. The province of Aceh has a prevalence of 1.4%, equivalent to the national average prevalence, according to a doctor’s diagnosis, namely 6,541 people [3].

Besides the threat of death, cancer pain is the most common source of disturbance associated with cancer. Even though for 50 years research has been conducted that claims cancer pain can be prevented and successfully reduced in 90% of cases, the majority of sufferers experience pain, with 33% reporting perceived pain intensity that is moderate or severe [1]. According to the research from the 2017 British Journal of Anesthesia, 1,564 (68%) of them reported pain [4].

The incidence of cancer is increasing in the older population as a vulnerable age group. Elderly patients diagnosed with cancer have many problems such as pain. It is estimated that 25%–40% of elderly individuals with cancer experience pain every day [5]. Measures of pain disturbance correlated with physical functioning, depression, and mood. Pain disorder is a predictor of patient quality of life [6]. In a Portuguese study, patients with more pain disorders and higher levels of pain intensity had worse physical functioning and more depression [5].

The main problem associated with the clinical management of cancer is controlling the accompanying pain by using various analgesics commonly used for this purpose. Recent evidence indicates that some analgesic targets, such as ion channels and receptors, are also involved in the cancer process, thus raising the possibility that the use of some of these analgesics may have an impact on cancer itself [7,8].

Gabapentin is one of the drugs used for the management of patients with cancer pain, especially neuropathic pain. There are studies showing the benefits and drawbacks of gabapentin in pain management in cancer. Previous research used a mouse model with PCA which was then treated daily with gabapentin 4.6–16.8 µg/kg by gavage. Primary tumorigenesis is monitored daily. Lung metastases were counted and measured after killing the mice 21 days later. Gabapentin has no effect on primary tumorigenesis, but it produces a dose-dependent effect on lung metastases [7].

The effect of systemic gabapentin administration on PCA was assessed for the first time. Gabapentin had no effect on the development of the primary tumor at all doses used. However, metastases to the lungs are affected in a dose-dependent manner with inhibition followed by augmentation of metastases. The multiple modes of action of gabapentin may be the reason why it can produce both pro- and antimetastatic effects [7].

Their study also showed that there was no effect on tumors at the lowest dose (4.6 µg/kg) tested, but metastases were reduced by 64% at 9.1 µg/kg. These observations suggest that gabapentin may have a selective inhibitory effect on the metastatic process. The antimetastatic effect of gabapentin may be due to voltage-gated sodium channel inhibition. However, at the highest dose (16.8 µg/kg) tested, gabapentin significantly increased the number of lung metastases by more than twofold [7].

Several methods of testing drugs as protection against cancer cell metastases can be carried out in vitro by testing the apoptotic index of these drugs to cancer cells. Testing the apoptotic index can be carried out by the flow cytometry method. The dose of gabapentin used for this in vitro test was based on research by Candotto et al. [9], namely 1,000 ng/mL, 2,000 ng/mL, and 4,000 ng/mL.

The research design used was an in vitro study with a randomized design. There were four groups in this study, namely four treatment groups and one control group with three repetitions per group. Gabapentin was given to each treatment group.

This research uses cervix cancer cell culture. The growing medium used was Ham’s F12 containing 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, and supplemented with 10% fetal bovine serum, and 1% penicillin-streptomycin. Cell multiplication is done by subculture through cell dissociation from the previous culture. The number of cells used in each treatment was counted using a hemocytometer.

Administration of gabapentin was diluted using distilled water and separated into three doses, 1,000 ng/mL, 2,000 ng/mL, and 4,000 ng/mL to find the optimal dose for cell apoptosis. Testing the apoptotic index can be carried out by the flow cytometry method.

Univariate analysis was performed by measuring the mean and standard deviation of each treatment. Bivariate analysis on the apoptotic variable used the one-way analysis of variance (ANOVA) test with a significant level (α) of 5% (0.05).

The apoptosis test was assessed in two stages, namely early and late apoptosis. The results of the gabapentin apoptotic index test on cancer cell cultures using the fl ow cytometry method can be seen in Table 1 below. The smallest average percentage of early apoptosis in cancer cell cultures was found in the control group, namely 3.07% ± 1.59%. The largest mean percentage of early cancer cell culture was found in the group given gabapentin 4,000 ng/mL, namely 30.50% ± 5.12%. The smallest average percentage of late apoptosis in cancer cell cultures was found in the control group, namely 1.47% ± 0.15%. The largest mean percentage of late apoptosis in cancer cell cultures was found in the group given gabapentin 4,000 ng/mL, namely 14.57% ± 1.86%.

Test results of early and late apoptosis showed that the smallest percentage of cancer cell culture apoptotic index was found in the control group that did not receive gabapentin therapy. While the highest percentage of cancer cell apoptosis index was found in the group given gabapentin with the largest dose of 4,000 ng/mL (Figure 1). The results of the ANOVA test showed that there was a signifi cant difference in the percentage of early and late apoptosis indices in the four groups based on the fl ow cytometry, with a P < 0.005.

Post hoc test results in Table 2 showed that there was a signifi cant difference in the percentage of apoptosis index between the control group and the group given gabapentin (P < 0.05) both in the early and late apoptosis. At the early apoptosis, there was a significant difference in the group given gabapentin 1,000 ng/mL, 2,000 ng/mL, and 4,000 ng/mL (P < 0.05). In the late apoptosis stage, the percentage of the apoptotic index of cancer cell cultures was not signifi cantly different in the control group, and the group given gabapentin 1,000 ng/mL; the group given gabapentin 1,000 ng/mL with 2,000 ng/mL (P > 0.05), and the group given gabapentin 2,000 ng/mL with 4,000 ng/ mL (P > 0.05) (Figures 1 and 2).

Table 1. Apoptosis Index Test Results

Figure 1. Graphic of Apoptosis Index Test Results

Table 2. Post Hoc Follow-Up Test Results for the Apoptotic Index Test in Each Group

Figure 2. Cancer Cell Culture Morphology After Gabapentin Treatment
(A) Control group, (B) Gabapentin 1,000 ng/mL, (C) Gabapentin 2,000 ng/mL, (D) Gabapentin 4,000 ng/mL.

Based on the research results, it was found that gabapentin was effective in increasing the apoptosis indices in cancer cell cultures. Several studies have reported that gabapentin apart from being an anti-neuropathic pain agent also has an antitumor effect in various models of cancer pain using experimental animals. The study by Brito et al. [10] shows that in addition to controlling mechanical and thermal hypersensitivity, gabapentin can also inhibit tumor proliferation, secretion of CC ligand 2, and calcium infl ux.

Gabapentin is an anticonvulsant drug that reduces synaptic transmission by lowering the presynaptic voltage of Ca2+ and Na+ channels. Gabapentin also reduces exocytosis and neurotransmitter release from presynaptic terminals [11]. Cell proliferation, which is a major component of the primary and secondary growth of cancer, involves a series of very complicated intracellular signals. The intracellular concentrations of Ca2+, H+ , Na+ , and K+ have all been shown to be important in determining cell cycle progression. Although the exact mechanism remains unclear, changes in ionic permeability contribute to the transition of cells from the G0, or early G1, quiescent state to the mitotic S phase [12].

Control of intracellular ion concentration is achieved through various membrane transport mechanisms including Na+ /K+ pumps, Na+ /H+ exchangers, Na+ /K+ /Cl− cotransporters, and IP3- induced intracellular Ca2+ release. It has been demonstrated that membrane ion channels play an important role in controlling cell growth and proliferation. It is known that cancer cells possess ion channels, that differential expression of ion channels can influence metastatic potential, and that ion channel expression can be controlled by mitogens and oncogenes [12].

Gabapentin, which is an adjuvant analgesic that is often used to treat neuropathic pain caused by cancer, is said to affect tumor development and progression in in vivo studies. Research that has been done using a mouse model with PCA and treated with gabapentin 4.6–16.8 µg/kg gavage shows that gabapentin has no effect on primary tumorigenesis, but produces a dose- dependent effect on lung metastases [7].

Gabapentin 16.8 µg/kg significantly increased metastasis by 112% and showed a strong tendency to shorten the mean survival time. They then concluded that gabapentin prescribed to cancer patients for pain might impact the cancer process itself [7].

Research by Bugan et al. [7] showed that there was no effect on tumors at the lowest dose (4.6 µg/kg) tested, but metastases were reduced by 64% at a dose of 9.1 µg/kg.

These observations suggest that gabapentin may have a selective inhibitory effect on the metastatic process. However, at the highest dose (16.8 µg/kg) tested, gabapentin significantly increased the number of lung metastases by more than twofold [7].

The effect of systemic gabapentin administration on PCA was assessed for the first time. Gabapentin had no effect on the development of the primary tumor at all doses used. However, metastases to the lungs are affected in a dose-dependent manner with inhibition followed by augmentation of metastases. The multiple modes of action of gabapentin may be the reason why it can produce both pro- and antimetastatic effects [7].

Survival of gabapentin-treated mice was reduced by approximately 16%. Gabapentin actually interferes with metastatic processes (i.e., tumor cells reach the lung) rather than proliferative activity (either in the primary tumor or metastases). The cause of the prometastatic effect of gabapentin was hitherto unclear at this time. Metastases generally involve changes in Ca2+. The effect of gabapentin on impaired Ca2+ in Mat-LyLu cells can produce a prometastatic effect [7].

Another study conducted at Kaiser Permanente Northern California showed no significant association between the use of gabapentin and the location of the cancer [13]. Epidemiological data in a US cohort with a follow-up of up to 12 years and a cohort in the UK with a follow-up of up to 15 years do not support a carcinogenic effect of gabapentin. However, the confidence intervals for some of the analyses are very wide, and significant effects cannot be ruled out with certainty [13].

Warnier et al. [14] assessed the additional subunit of the α2δ2 calcium channel (CACNA2D2 gene) which is thought to be involved in the development of PCA. This study assessed the expression of proteins in LNCaP PCA cells and in PCA tissue that can be altered during cancer development and the influence of overexpressing or downregulating this subunit on cell proliferation. In vitro experiments showed that overexpression of the α2δ2 subunit was associated with increased cell proliferation, changes in calcium homeostasis, and activation of T-cell pathways. In vivo studies were conducted in immunodeficient mice to evaluate the tumorigenic potential of the α2δ2 subunit [14].

LNCaP PCA cells with α2δ2 overexpression were more tumorigenic compared to control LNCaP cells when injected into these mice. Gabapentin, which is a ligand of α2δ2, reduces tumor development in xenografts LNCaP. This study shows that the action of α2δ2 on tumor development occurs not only through stimulation of proliferation but also through stimulation of angiogenesiswith increased secretion of vascular endothelial growth factor in cells that overexpress [14].

Gabapentin is a derivative of the gamma-aminobutyric acid (GABA)-inhibitory neurotransmitter originally designed as a GABA mimetic agent licensed to treat refractory epilepsy. Gabapentin has gained wide acclaim for its efficacy in controlling chronic pain syndromes, especially neuropathic pain, with minimal side effects. Several studies have demonstrated specific binding to gabapentin at the Cavα2δ subunit that stimulates the characterization of voltage-gated calcium channels (VGCCs) in the clinical activity of the drug. Current evidence supports the existence of four isoforms of the Cavα2δ subunit, only two of which (Cavα2δ1 and Cavα2δ2) bind gabapentin at L-, N- and P/Q-type calcium channels. This binding promotes inhibition of calcium inflow and consequently leads to reduced release of glutamate and other excitatory molecules such as substance P. Gabapentin-induced reduction of glutamate release may modulate metabotropic glutamate receptor 1 occupancy, as well as N-methyl-D-aspartate (NMDA) receptors. Glutamate, acting via the NMDA receptor, plays a role in altering natural killer cell responses, which are endogenously responsible for reducing tumor activity [10].

Brito et al. [10] reported that expression of VGCCs has been reported in melanoma cells, which play a role in tumorigenesis and tumor development. Aberrant expression and abnormal activity of specific ion channels are associated with increased cancer aggressiveness. Therefore, pharmacological regulation of channel activity may offer protection against some types of cancer. Melanoma cells express channel isoforms belonging to caveolin 1 (Cav1) (which encodes mainly high-voltage-activated L-type channels) and Cav2 (which encodes high-voltage-activated Q-type, N-type, and R-type channels). Therefore, agents that modulate these calcium channels such as gabapentin may be a therapeutic option to prevent proliferation, migration, or invasion of cancer cells and concurrently as a therapy for neuropathic pain due to cancer [10].

Administration of gabapentin at doses of 1,000 ng/mL, 2,000 ng/mL, and 4,000 ng/mL was effective in increasing the apoptotic index of cancer cell cultures.