Background
Cancer is a major public health problem that is currently one of the most leading causes of death in the United States. The cancer death rate escalated during the 20th century which was largely driven by rapid increases in lung cancer deaths as a consequence of tobacco epidemic. In 2015, the estimated number of cancer deaths in USA is dominated by lung cancer (27 % of all cancer deaths), surpassing prostate cancer for males (9 %) and breast cancer for females (15 %) [
1]. In South Korea, meanwhile, lung cancer is expected to contribute 12,736 deaths in men, the highest amount among all cancers [
2]. This increasing mortality rate in lung cancer patients has led physicians and scientists to seek a deeper understanding on this malignancy.
Lung cancer is a type of cancer that initially forms in lungs and can spread to nearby tissue even other organs, with the most recurrent metastatic sites were nervous system, bone, liver, respiratory system and adrenal gland [
3]. Several risk factors may increase the chance to acquiring lung cancer, with mostly related to exogenous compounds found in cigarette smoke and synthetic manufacturing materials [
4]. In fact, nine out of ten lung cancer cases are caused by smoking cigarettes [
5]. Moreover, smokers have a greater risk for lung cancer today than they did in 1964, even though they smoke fewer cigarettes.
There are several diagnostic tools that are available in detecting lung cancer such as computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET) [
6‐
8]. However, lung cancer patients do not experience signs and symptoms at the early stages of the disease which lead to late diagnosis—often when the cancer has already progressed. Furthermore, there are no initial screening methods available to diagnose lung cancer at its early stages.
A study mentioned that early detection of lung cancer can be performed by NMR-based metabolomics method for urine samples [
9]. But the changes in metabolite levels in urine still cannot be correlated to compounds involved in lung cancer metabolism. Furthermore, metabolite levels in urine samples may vary depending on fluid intake prior to collection, and may also be affected by patients’ kidney function [
10]. Miyamoto et al. (2015) conducted plasma samples analysis using GC-MS in determining metabolic changes of endogenous compounds in lung cancer patients [
11]. However, the detection of exogenous compounds were not considered. Some other researches have dealt with respiratory diseases using NMR metabolomics like the study on electronic nose and exhaled breath [
12], and on cystic fibrosis [
13].
During the past decade, liquid chromatography coupled with mass spectrometry (LC-MS) has gone through huge development. The ability to determine thermolabile compounds directly without needing to undergo derivatization steps has given its advantage over gas chromatography (GC). With recently-developed configuration like quadrupole time-of-flight (Q-TOF), the range of screening has quickly expanded, enhancing both high mass resolution and mass fragmentation [
14,
15].
Due to the disadvantages of urine samples mentioned earlier, this study aims to explore and identify significant compounds, exogenous and endogenous, that may induce cancer using LC-MS based high resolution metabolomics (HRM) on human serum samples from South Korean males for the purpose of providing early detection and non-invasive diagnosis of lung cancer. The significant compounds found to contribute in the discrimination between healthy and lung cancer subjects will also be confirmed and their correlation with smoking habit will be examined.
Discussion
This study explored lung cancer-specific low molecular weight compounds that may have potential roles in inducing the disease and can be used as candidate biomarkers for the early diagnosis of lung cancer. Using serum samples from all patients, three compounds: BPA, retinol, and L-proline were identified as statistically significant compounds and were correlated to the subjects’ smoking habit.
BPA is used in the production of polycarbonate plastics which is the main ingredient of many manufactured goods such as toys, drinking containers, food cans, eyeglass lenses and electronic appliances [
27‐
29]. It was found to be significant only in past smoker control vs. past smoker LCPs analysis (as shown in Additional file
9). With this finding, it is possible that this compound may induce lung cancer but it is not related to the smoking history of the subjects. Since it was detected insignificant under non-smoker control vs. current-smoker LCPs analysis group, it can be inferred that BPA may have come from other sources (e.g. plastic products, food cans) and not from cigarettes. Previous studies have mentioned several possible roles of BPA in carcinogenesis [
30‐
32]. Although having a BPA concentration of less than 10
−4 M had not triggered the proliferation process of lung cancer cells, it had stimulated in vitro migration and invasion of the cells via up-regulation of matrix metalloproteinase-2 which could enhance the susceptibility to carcinogenesis. Another study showed that BPA could alter Peroxisome Proliferator-activated Receptors (PPARs), in this case, the PPARγ. This ligand-activated transcription factor was found to induce differentiation and apoptosis in lung cancer cells. As its antagonist, BPA promoted prevention of apoptosis, resulting the survival of cancer cells [
30‐
32]. In addition, BPA concentration in human samples was correlated with intracranial tumor [
33] and prostate cancer [
34].
Vitamin A, on one hand, is also one of the selected metabolites observed to be differentially expressed in the comparison groups. This vitamin comes from carotenoid and retinoid. Basically, carotenoids are known for their antioxidant effects and are derived from plants [
35]. Retinoid are physiological regulators of embryonic development, vision, reproduction, bone formation, hematopoiesis, differentiation, proliferation and apoptosis [
36]. Retinol was found to be effective against breast, prostate, and ovarian cancers in animal treatment models. Also, the ability of retinol to induce apoptosis suggested that it is potential in prevention and treatment of lung cancer and other types of cancer [
37]. In this study, detection of retinol was significantly less in current-smoker patients as compared to non-smoker control (as shown in Additional file
9). Therefore, it is suggested that the level of retinol in the body may have been decreased in patients who smoke, thus increasing their risks in having the disease. In addition, using control and LCPs comparison, KEGG mapping displayed vitamin digestion and absorption as one of the affected human pathway. This finding was consistent with previous reports. Cigarette smoke could induce depletion of retinol serum levels in rats, as demonstrated by Li et al. [
38]. It was further investigated to be correlated with the development of emphysema. In addition, Yuan et al. revealed that low level of serum retinol is associated with the increase of lung cancer risk in China population [
39].
Meanwhile, L-proline, a non-essential amino acid, was found to be lowered in LCPs, confirming a previously reported study [
40]. Zhao et al. reported that L-proline was one of the amino acid which its plasma concentration was found to be decreased in LCPs, compared with control group (
p < 0.001). Rapid increase in the transcription of proline dehydrogenase by tumor suppressor p53 triggered the degradation of this amino acid in cancer [
41]. In addition, using control and LCPs comparison, KEGG mapping displayed biosynthesis of amino acids as one of the affected human pathway. As shown in Additional file
9, the decreasing L-proline occurred in all parameters that were analyzed, suggesting that this compound is possible to act as biomarker for lung cancer, regardless of smoking habit.
Conclusions
In summary, two potential biomarkers, retinol and L-proline, were identified using HRM with the combination of pathway analysis from significant metabolites that were found in serum samples of Korean male LCPs. It was also seen that one of them was correlated to smoking habit of LCPs. In addition, one exogenous compound, BPA, which has not been linked yet to lung cancer patients was demonstrated as significant feature that contributed to discriminate healthy and LCPs groups, regardless of smoking habit. These findings may create opportunities for the development of new lung cancer diagnostic tools. In the future, correlations to disease type, stage, also the applicability to female test subjects group may also be conducted. Also, further study using larger population should be conducted.
Abbreviations
BPA, bisphenol A; FDR, false discovery rate; HCA, hierarchical cluster analysis; HRM, high resolution metabolomics; KEGG, kyoto encyclopedia of genes and genomes; LCPs, lung cancer patients; Q-TOF, quadrupole time-of-flight
Acknowledgements
The authors thank Ryan De Sotto and Carl Medriano for providing different insights and comments for the manuscript. ADP gratefully acknowledges Indonesia Endowment Fund for Education (LPDP) for the financial support of his master degree scholarship.