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Original article
Generation of gaseous sulfur-containing compounds in tumour tissue and suppression of gas diffusion as an antitumour treatment
  1. Kazue Yamagishi1,
  2. Kazuo Onuma1,
  3. Yota Chiba1,
  4. Shinya Yagi2,
  5. Shigenobu Aoki3,
  6. Tomoyuki Sato4,
  7. Yasushi Sugawara5,
  8. Noriyasu Hosoya6,
  9. Yasutake Saeki7,
  10. Minoru Takahashi8,
  11. Masayoshi Fuji8,
  12. Takeo Ohsaka9,
  13. Takeyoshi Okajima9,
  14. Kenji Akita10,
  15. Takashi Suzuki11,
  16. Pisol Senawongse12,
  17. Akio Urushiyama13,
  18. Kiyoshi Kawai14,
  19. Hirofumi Shoun15,
  20. Yoshimasa Ishii16,
  21. Hiroya Ishikawa17,
  22. Shigeru Sugiyama18,
  23. Madoka Nakajima19,
  24. Masaru Tsuboi19,
  25. Tateo Yamanaka20
  1. 1FAP Dental Institute, Tokyo, Japan
  2. 2Department of Quantum Engineering, Graduate School of Engineering, Nagoya University, Nagoya, Aichi, Japan
  3. 3Faculty of Social and Information Studies, Gunma University, Maebashi, Gunma, Japan
  4. 4Center of Colorectal Disease and Pelvic Floor Dysfunction, Saitama Shinkaibashi Clinic, Saitama city, Saitama, Japan
  5. 5Department of Plastic Surgery, Jichi Medical University, Shimotsuke, Tochigi, Japan
  6. 6Department of Periodontics and Endodontics, Tsurumi University, School of Dental Medicine, Yokohama, Kanagawa, Japan
  7. 7Department of Physiology, Tsurumi University, School of Dental Medicine, Yokohama, Kanagawa, Japan
  8. 8Nagoya Institute of Technology, Tajimi, Gifu, Japan
  9. 9Department of Electrical Chemistry, Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology, Yokohama, Kanagawa, Japan
  10. 10Department of Respiratory Medicine, Nagoya Central Hospital, Nagoya, Aichi, Japan
  11. 11Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Yamanashi University, Kofu, Yamanashi, Japan
  12. 12Department of Operative Dentistry, Faculty of Dentistry, Mahidol University, Bangkok, Thailand
  13. 13Department of Chemistry, College of Science, St. Paul's (Rikkyo) University, Tokyo, Japan
  14. 14Department of Nutrition, Faculty of Wellness, Shigakkan University, Ohbu, Aichi, Japan
  15. 15Department of Biotechnology, Graduate School of Agricultural and Life Science, University of Tokyo, Tokyo, Japan
  16. 16Dentistry/Oral and Maxillofacial Surgery, Ebina General Hospital, 1320 Kawaraguchi, Ebina, Kanagawa, Japan
  17. 17Department of Nutrition and Health Science, Faculty of Human Environmental Science, Fukuoka Women's University, Fukuoka, Japan
  18. 18Graduate School of Engineering, Osaka University, Suita, Osaka, Japan
  19. 19Bio-Safty Research Center, Foods, Drugs, and Pesticides (BSRC), Iwata, Shizuoka, Japan
  20. 20Tokyo Institute of Technology, Kochi, Japan
  1. Correspondence to Dr Kazue Yamagishi, FAP Dental Institute, 3-2-1, Kakinokizaka, 502, Meguro-ku, Tokyo 152-0022, Japan; fzt02705{at}nifty.com

Abstract

Background and aims The mechanisms of cancer cell growth and metastasis are still not entirely understood, especially from the viewpoint of chemical reactions in tumours. Glycolytic metabolism is markedly accelerated in cancer cells, causing the accumulation of glucose (a reducing sugar) and methionine (an amino acid), which can non-enzymatically react and form carcinogenic substances. There is speculation that this reaction produces gaseous sulfur-containing compounds in tumour tissue. The aims of this study were to clarify the products in tumour and to investigate their effect on tumour proliferation.

Methods Products formed in the reaction between glucose and methionine or its metabolites were analysed in vitro using gas chromatography. Flatus samples from patients with colon cancer and exhaled air samples from patients with lung cancer were analysed using near-edge x-ray fine adsorption structure spectroscopy and compared with those from healthy individuals. The tumour proliferation rates of mice into which HT29 human colon cancer cells had been implanted were compared with those of mice in which the cancer cells were surrounded by sodium hyaluronate gel to prevent diffusion of gaseous material into the healthy cells.

Results Gaseous sulfur-containing compounds such as methanethiol and hydrogen sulfide were produced when glucose was allowed to react with methionine or its metabolites homocysteine or cysteine. Near-edge x-ray fine adsorption structure spectroscopy showed that the concentrations of sulfur-containing compounds in the samples of flatus from patients with colon cancer and in the samples of exhaled air from patients with lung cancer were significantly higher than in those from healthy individuals. Animal experiments showed that preventing the diffusion of sulfur-containing compounds had a pronounced antitumour effect.

Conclusions Gaseous sulfur-containing compounds are the main products in tumours and preventing the diffusion of these compounds reduces the tumour proliferation rate, which suggests the possibility of a new approach to cancer treatment.

  • Hydrogen sulfide
  • glucose metabolism
  • colorectal cancer
  • cancer
  • oncogenes
  • adhesion molecules
  • mutations
  • gene mutation
  • genetic testing
  • gut immunology
  • gut inflammation
  • growth factors
  • gut differentiation
  • gut hormones

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Significance of this study

What is already known about this subject?

  • Glycolytic metabolism is markedly accelerated in cancer cells, causing the accumulation of glucose (a reducing sugar) and methionine (an amino acid) which are used as indicators in cancer screening using positron emission tomography.

  • The reaction between reducing sugars and amino acids can produce carcinogenetic materials such as acrylamide.

  • Lactic acid is produced as a metabolic product in cancer cells, and an increase in its level in blood is strongly related to cancer progression.

  • Cancer patients produce certain odorous compounds and dogs can detect them by smell. This can be used as a new diagnostic approach to cancer detection.

  • Sulfur ions in hydrogen sulfide gas bond the iron atoms in haemoglobin, which interferes with the function of cytochrome C oxidase.

What are the new findings?

  • Glucose and methionine or its metabolites accumulate in cancer cells causing a Maillard reaction which produces gaseous sulfur-containing compounds such as methanethiol and hydrogen sulfide.

  • Lactic acid, which is a product in cancer cells, and sulfur-containing amino acid produce methanethiol and hydrogen sulfide.

  • The amount of methanethiol in flatus from patients with colon cancer and the amount of hydrogen sulfide in exhaled air from patients with lung cancer is significantly higher than that from healthy individuals.

  • Animal experiments showed that prevention of diffusion of sulfur-containing gases in tumour caused a significant decrease in the tumour proliferation rate.

  • The binding of sulfur atoms in hydrogen sulfide gas and of iron atoms in haemoglobin occurs in the presence of glucose.

How might they affect clinical practice in the foreseeable future?

  • Sulfur-containing gases are highly sensitive indicators for colon and lung cancer screening, and the amount of these gases indicates whether the detoxification system is working correctly, especially in the large intestine.

  • Preventing diffusion of sulfur-containing gas in tumour tissues could be an efficient approach to preventing cancer invasion.

Introduction

Cancer not only forms localised lesions but can also be considered a systemic disease that impairs the immune functions by causing cell proliferation and metastasis.1 2 Consequently, modern cancer treatments vary widely and, in addition to the conventional approach of surgically removing the tumour tissue or using anticancer drugs,3 4 other approaches such as immunotherapy5 and gene therapy are being tested.6 7 Although the mechanisms of cancer cell growth and metastasis are still not entirely understood, the complex phenomena induced by tumour tissue should be expressible as the result of various chemical reactions,8–10 and it can be conjectured that the basic reactions and their products are strongly related to the cancer pathology.9 11–14

Substances that are widely recognised to be among those that accumulate in tumour tissue are glucose (a reducing sugar)15 and methionine (a sulphur-containing amino acid).8 16 Reducing sugars and amino compounds can undergo a non-enzymatic Maillard reaction, which produces various substances and is linked with carcinogenic activity at normal body temperature in a neutral pH environment.17 18

Previous research results suggest that the Maillard reaction takes place between glucose and methionine in tumour tissue and that the reaction products are linked to cancer pathology.19 This reaction also takes place in vivo between glucose and homocysteine or cysteine, which are amino acid metabolites of methionine. However, there are no reports of results confirming this. To obtain such results, it is necessary to identify the reaction products. We focused on the sulfur atoms in methionine, homocysteine and cysteine and investigated the production of gaseous sulfur-containing compounds from glucose and these amino acids. Some sulfur-containing compounds, including hydrogen sulfide gas, adversely affect living organisms, sometimes fatally.20–25 The generation of gaseous sulfur-containing compounds in tumour tissue and their diffusion inside the body may suppress aerobic metabolism in the surrounding healthy cells,21 causing the creation of an anaerobic environment that leads to cancer proliferation and metastasis.26 Thus, improvement of the hypoxic condition by reducing these compounds could be a new approach to tumour treatment.

Materials and methods

Procedure for in vitro Maillard reaction and gas chromatography

Mixtures of D-glucose (I'ROM Co Ltd, Atsugi, Japan) with L- and D-methionine, L-homocysteine and L-cysteine (Wako Co Ltd, Osaka, Japan) were used for the experiments. Each mixed solution was placed in a glass bottle (inner volume 50 ml), which was then sealed and kept at 25°C for 3 days. The gaseous materials produced by the reaction in the solution and that concentrated in the free space above the solution were then gathered over a 24-h period using a Tenax tube with flowing nitrogen gas at 37°C and sent to a gas chromatographer (Shimadzu GC2014). The capillary column (CP-SilicaPLOT) in the gas chromatographer was 60 m long with an inner diameter of 0.53 mm. Helium was flowed as a carrier gas at 8 ml/min. The column was kept at 60°C for 3 min and then the temperature was increased to 180°C at 10°C/min for measurement. The detector was a flame photometric type and the gas detection temperature was 200°C.

Preparation of rhodium nanoparticle-coated plate and samples for near-edge x-ray fine adsorption structure spectroscopy (NEXAFS)

Nickel plates (10×10×0.5 mm) coated with rhodium nanoparticles (average diameter 3.0 nm) were used to detect the sulfur-containing compounds in flatus and exhaled air samples. They were prepared by dissolving rhodium nanoparticles in a 90 v/v% ethanol-aqueous solution with a concentration of 0.2 wt%. A 100 μl portion of this solution was spin coated at 4000 rpm onto nickel plates, forming a rhodium nanoparticle layer. The plates were then heated at 300°C for 2 min in air. A prepared plate was directly exposed to flatus or exhaled air for near-edge x-ray fine adsorption structure spectroscopy (NEXAFS) measurement (BL-3, Synchrotron Radiation Center, Hiroshima University).

Resonance Raman spectroscopy measurement

Peripheral blood taken from a healthy individual (control) was compared with the same blood to which hydrogen sulfide gas has been added (control + H2S) and with the same blood to which hydrogen sulfide and glucose had been added (control + H2S + glucose). The blood volume for measurement was 1 ml. Ten μl pure water in which hydrogen sulfide gas had been bubbled for 10 min (pH of 3.9) was added to the control sample in preparing the control + H2S sample and 100 μl 50% D-glucose solution was added to the control + H2S sample in preparing the control + H2S + glucose sample.

Animal experiments for investigating sulfur-containing gas diffusion into body from tumour

Six-week-old female nude mice (BALB/c nu/nu, Japan SLC Co Ltd) were used for the animal experiments. The mice used for the measurement of the tumour proliferation rate were prepared by injecting approximately 1×107 HT29 human colon cancer cells into their flanks. They were then placed into one of two groups (five per group) and further prepared: (1) control mice (no further treatment); (2) mice injected with a gel solution of sodium hyaluronate (Na-HA) around the outside of the tumour. The Na-HA was prepared by dissolving hyaluronic acid in distilled water at a concentration of 10 mg/ml. The pH of the mixed gel solution was adjusted to around 7.0–7.5 using NaH2PO4 and Na2HPO4. As an isotonic agent, NaCl was added to this solution at a concentration of 152 mM. The chemical formula for the Na-HA was (C14H20NNaO11)n and the average polymer molecular weight was approximately 3000 kDa. A 0.2 ml portion of the Na-HA was injected subcutaneously around the tumour every 3 days. All animal experiments were done in a class-1000 clean room.

Calculation of tumour proliferation rate and preparation of sample for NEXAFS measurement

The tumour proliferation rate was calculated by estimating the change in tumour area over time through the skin. After 28 days of mouse growth, the tumours in the control group mice were extracted. The centre and peripheral regions of the tumour were separately immersed in 80 v/v% ethanol-aqueous solution with a rhodium nanoparticle-coated substrate to adsorb the sulfur-containing compounds for NEXAFS measurement.

Preparation of samples for transmission electron microscopy (TEM) observation

Blood plasma was separated from peripheral blood taken from patients with lung cancer. Three types of solutions were prepared: (1) Na-HA gel solution (control sample); (2) 1 ml Na-HA dissolved in 1 ml plasma; and (3) solution prepared by immersing a rhodium nanoparticle-coated substrate wrapped in a semipermeable membrane (Spectrum Laboratories Inc, Dallas, USA MWCO: 6–8 kDa) in plasma for 12 h to adsorb the gaseous sulfur-containing compounds and then 1 ml of this plasma was mixed with 1 ml Na-HA. The concentration of the Na-HA in these three solutions was 10 mg/ml. After each sample was diluted 100 times using pure water, approximately 100 μl of the resulting solution was placed on a platinum grid and negatively stained using uranyl acetate solution for TEM (JEOL, JEM2000EX) observation.

Results

In vitro Maillard reaction

Figure 1 shows gas chromatography spectra for the products of in vitro reactions between glucose and methionine, glucose and homocysteine, and glucose and cysteine. Each amino acid was dissolved in a 50% aqueous D-glucose solution at a concentration of 30 mg/ml (case 1: pink spectra) or dissolved in a 25% aqueous D-glucose solution which was prepared by diluting 50% glucose with physiological saline at a concentration of 3 mg/ml (case 2: blue spectra). The mixed solutions were left for 3 days at 25°C and the gases in the sealed spaces above the liquid were then measured. For L-methionine (figure 1A), 0.36 ppm methanethiol was detected as a reaction product under the conditions of case 1. Since the lower detection limit of methanethiol by gas chromatography is approximately 0.05 ppm, this value is significant. Under the conditions of case 2, the methanethiol concentration was below the detection limit. Naturally occurring methionine is laevorotatory but, interestingly, when reaction experiments were performed using D-methionine, there was an almost eightfold increase in the yield of methanethiol (2.92 ppm) under the conditions of case 1 (figure 1B). Dimethyl disulfide (0.06 ppm) was also detected: it was considered to be a product of polymerisation between methanethiol molecules. For the D-methionine, 0.24 ppm methanethiol was detected even under the conditions of case 2. This means that the generation of methanethiol is greatly accelerated by the production of D-methionine, which is of no benefit to living organisms.

Figure 1

Gas chromatography spectra for in vitro Maillard reaction products. Pink spectra correspond to conditions of case 1 and blue spectra correspond to those of case 2. Reaction between (A) L-methionine and D-glucose; (B) D-methionine and D-glucose; (C) L-homocysteine and D-glucose; and (D) L-cysteine and D-glucose. Peaks around 1 min in all spectra correspond to an artefact resulting from pressure fluctuation of the carrier gas.

The spectra for the L-methionine metabolites L-homocysteine and L-cysteine are shown in figure 1C and D, respectively. Under the conditions of case 1, hydrogen sulfide (91.1 ppm) and methanethiol (1.33 ppm) were detected with homocysteine, and hydrogen sulfide (387 ppm), carbonyl sulfide (4.40 ppm), carbon disulfide (0.34 ppm) and methanethiol (0.23 ppm) were detected with cysteine. Under the conditions of case 2, only hydrogen sulfide was detected with both homocysteine (4.94 ppm) and cysteine (74.1 ppm). The production of hydrogen sulfide at a high concentration was characteristic for both amino acids but not for methionine.

These results demonstrate that, when sulfur-containing amino acids (such as methionine and its metabolites homocysteine and cysteine) are present with glucose, the Maillard reaction can occur even at normal temperatures, producing various gaseous sulfur-containing compounds.

Lactic acid accumulates in tumour tissue through transformation of pyruvic acid by lactate dehydrogenase. We found that gaseous sulfur-containing compounds were also produced by the reaction between lactic acid and methionine (see figure 1 in online supplement).

We also found that bubbling of cigarette smoke—a high risk factor for lung cancer—into glucose solution produced approximately four times the methanethiol produced by bubbling it into simple saline solution (see figure 2 in online supplement).

NEXAFS of flatus and exhaled air from cancer patients

We investigated whether the gaseous sulfur-containing compounds that have been shown in vitro to be produced from the combination of glucose and methionine (and the methionine metabolites homocysteine and cysteine) are actually produced in human tumour tissue. It is thought that some of these gaseous compounds are absorbed in the body while others are expelled. To detect trace quantities of sulfur-containing compounds diluted in gases emitted from the body, we obtained samples of flatus from patients with colon cancer and exhaled air from patients with lung cancer and used NEXAFS to identify and quantify their constituents. The samples were directly exposed to a rhodium nanoparticle-coated substrate that selectively adsorbs molecules with -CSH bonds (such as methanethiol). Schematic illustrations of the methods used to collect the samples are shown in figure 3 in the online supplement.

Figure 2A shows the NEXAFS reference spectra used to identify the peak positions corresponding to bonds involving sulfur atoms. These spectra correspond to a thin film substrate with rhodium nanoparticles deposited on it (orange), a rhodium single crystal (100) substrate on which hydrogen sulfide gas (containing sulfur atoms) was adsorbed (light brown) and a rhodium single crystal substrate that had been dipped in a solution of L-cysteine (including -SH thiol groups) (aqua). The peak at 2482.0 eV in the spectrum for the rhodium nanoparticle substrate corresponds to sulfur oxide (mainly sulfates), which was an impurity in the substrate. The peak at 2470.6 eV in the spectrum for the rhodium single crystal substrate with adsorbed hydrogen sulfide gas corresponds to the bonds between the rhodium and sulfur atoms. The peak at 2472.8 eV for the rhodium single crystal substrate dipped in an L-cysteine solution corresponds to bonds between the rhodium atoms and -CSH groups. This is because the -SH bonds in the adsorbed L-cysteine were very weak so they easily changed into -CSH bonds at room temperature. This peak can be used as a reference point for the detection of methanethiol.

Figure 2

Near-edge x-ray fine adsorption structure spectroscopy (NEXAFS) measurements for detecting gaseous sulfur-containing compounds. (A) Reference spectra for screening flatus samples. (B) Average spectra of flatus samples from colon cancer (red), healthy (blue) and silky egg (light brown) groups. Inset panel is enlarged spectra up to 2480 eV. Vertical dotted line, horizontal line and double arrows indicate position of Rh-CSH component peak, base line used for calculating peak intensity and peak intensity for cancer group, respectively. (C) Comparison of methanethiol (Rh-CSH component) content in flatus for colon cancer, healthy and silky egg groups. Data for cancer group are plotted on the basis of tumour progression (Dukes' classification). (D) Spectra of exhaled air from patient with lung cancer (red) and healthy individual (blue).

In measurements using rhodium single crystal substrates the peak at 2482.0 eV corresponding to sulfur oxide impurities was not observed when the substrate was carefully purified. However, since this impurity peak does not interfere with the peaks corresponding to bonds between the rhodium and sulfur atoms or between the rhodium atoms and -CSH groups, the rhodium nanoparticle-coated substrates used for the adsorption of flatus and exhaled air were not purified. The small wide peak near 2477 eV, seen when using the rhodium single crystal substrate, was the result of multiple scattering due to bonds between the rhodium and sulfur (atoms or moieties).

Figure 2B compares the NEXAFS spectra of flatus from healthy individuals and patients with colon cancer. To account for dietary-related differences in the sulfur-containing compounds in the flatus, we also analysed the flatus of healthy individuals who had eaten silky eggs. For the patients with colon cancer we obtained an average spectrum for 20 individuals whose cancer had progressed to differing degrees and, for the healthy individuals, we obtained an average spectrum for 15 individuals and for four individuals who had eaten silky eggs. In the average spectrum for the patients with colon cancer the peak at 2472.8 eV was stronger than in the spectra for the other two groups, suggesting that methanethiol was generated in the tumour tissue. There was no peak at 2470.6 eV corresponding to bonds between the rhodium and sulfur atoms.

We investigated whether the results plotted in figure 2B are statistically significant. Figure 2C compares the methanethiol content (2472.8 eV peak height) in the samples for all the spectra plotted in figure 2B. For the patients with colon cancer the state of progress was classified from A to D in accordance with the Dukes' staging system.27 We applied a homogeneity of variance t test to the raw data obtained for the colon cancer and healthy groups and obtained F=837.3, first degree of freedom=19.0, second degree of freedom=14.0 and p=2.2×10−6. Since both groups were clearly heterogeneous with regard to variance, we used an unpaired t test (Welch's t test) to investigate the difference in methanethiol content. This yielded t=2.8, degree of freedom=19.1 and p=1.1×10−2. Furthermore, since the data from the two groups do not conform to a normal distribution, we performed a Wilcoxon rank sum test and obtained W=300 and p=6.2×10−7. These statistical results show that the methanethiol content was clearly increased in the flatus of the patients with colon cancer. The number of data points for the silky egg group was much smaller than for the other two groups, so statistical analysis was not done for the silky egg group.

NEXAFS spectra for a patient with lung cancer (small cell carcinoma, stage IIIb) and a healthy individual are shown in figure 2D. Both the hydrogen sulfide (2470.6 eV) and the methanethiol (2472.8 eV) peaks were intense for the cancer patient. Detection of a 2470.6 eV peak for the exhaled air sample but not for the flatus sample for the cancer patient suggests that biocatalysis (such as bacteria and enzyme activity) in the intestines preferentially acts on the metabolism of hydrogen sulfide.

Inhibition of gas diffusion: animal tests

The findings from the in vitro demonstration of the Maillard reaction between glucose and sulfur-containing amino acids and from the NEXAFS analysis of flatus and exhaled air samples from patients with cancer suggest that the generation of gaseous sulfur-containing compounds in tumour tissue contributes substantially to cancer proliferation and metastasis. We verified this by conducting animal experiments to determine if preventing the generation and diffusion of sulfur-containing compounds has any antitumour effects.

We measured the change in tumour area over time for mice into which 1×107 HT29 colon cancer cells had been transplanted. The groups were (1) an untreated (control) group and (2) a group in which Na-HA gel solution was injected around the outside of the tumour tissue to form a layer trapping the gaseous sulfur-containing compounds. The tumour tissue for the control group was excised to confirm the existence of a gaseous sulfur-containing compound by NEXAFS on the 28th day after treatment.

In the NEXAFS measurement results for the excised tumour tissue for the control group we observed a hydrogen sulfide peak at 2470.6 eV and a methanethiol peak at 2472.8 eV (figure 3A). Comparison of the quantity of sulfur-containing compounds at the centre and periphery of the tumour tissue showed that there was more methanethiol than hydrogen sulfide at both the centre and periphery (figure 3B). An unpaired t test showed p values of 0.016 for the centre and 0.033 for the periphery, indicating that the differences between the quantities of methanethiol and hydrogen sulfide were statistically significant.

Figure 3

Results of animal experiments to investigate tumour proliferation rate and effect of antitumour treatments using HT29 human colon cancer cells. The Institutional Animal Care and Use Committee at Takara Bio Inc. approved all mouse-handling procedures. (A) Average near-edge x-ray fine adsorption structure spectroscopy (NEXAFS) spectra for tissue taken from tumour centre (red) and periphery (blue) of control mice. Hydrogen sulfide (2470.6 eV peak) and methanethiol (2472.8 eV peak) were detected in both. (B) Amount of hydrogen sulfide (grey bar) and methanethiol (flesh-coloured bar) at tumour centre and periphery. (C) Change in tumour area over time for each mouse in the control group. Different symbols correspond to data for different mice. (D) Change in tumour area over time for each mouse in sodium hyaluronate (Na-HA) injected group. (E) Results of t test for tumour proliferation rate between control and Na-HA injected groups.

Sulfur-containing compounds such as hydrogen sulfide are a cytotoxin which inhibits cytochrome C oxidase,21 and their sulfur ions bond to Fe at heme active sites in haemoglobin, blocking the transport of oxygen in the blood. Resonance Raman spectroscopy measurements of blood samples taken from healthy individuals to which hydrogen sulfide gas had been added revealed that Fe-S bonds form much more readily in the presence of glucose (figure 4). This reaction places cells in an anaerobic environment, where it is thought that the acceleration of glycolytic metabolism28 promotes the infiltration of cancer, leading to proliferation and metastasis. Thus, preventing the generation and diffusion of gaseous sulfur-containing compounds should reduce the adverse effects on cells in the vicinity of the tumour and also systemically, thereby increasing the efficacy of antitumour treatments.

Figure 4

Resonance Raman spectra of peripheral blood taken from a healthy individual (control), the same blood to which hydrogen sulfide gas had been added (control + H2S) and the same blood to which hydrogen sulfide and glucose had been added (control + H2S + glucose). When hydrogen sulfide was added, a small shoulder peak (orange arrow) appeared at around 453 cm−1, adjacent to the peak at around 430 cm−1 (dark blue arrow). The intensity of the 453 cm−1 peak became comparable to that of the 430 cm−1 peak when glucose was added. The 453 cm−1 peak corresponds to Fe-S stretching band. Comparison between control spectrum and the other two spectra showed that no new peak except that at 453 cm−1 appeared when H2S and glucose were added, although the relative peak intensities (especially in high wave number regions) were different. This means that a change in the spectrum reflected the occurrence of a chemical reaction in the heme colour centre and did not correspond to a change in the heme framework. The combination of hydrogen sulfide and glucose accelerated the formation of Fe-S bonds in the heme.

Figure 3C and D show the change in tumour area over time for each mouse in the control group and in the Na-HA injected group. Of the 10 measurements, one sample from the Na-HA injected group had a correlation coefficient of a linear function (0.97) and the others all had a correlation coefficient >0.98. The average tumour proliferation rates and their SDs were 5.7±1.2 mm2/day for the control group and 4.0±0.7 mm2/day for the Na-HA injected group. The reduction in average tumour proliferation rates compared with the control group was approximately 30% for the Na-HA injected group.

Figure 3E shows the results of an unpaired t test to determine whether the difference in the proliferation rate between the control and Na-HA injected groups was significant. A significant difference was found between the groups (p=0.015), which means that the Na-HA trap had a strong antitumour effect.

Animal experiments using HeLa S3 cancer cells were also done to determine whether the findings obtained using HT29 cells were specific to the kind of tumour cell. The results were essentially the same as those for HT29 cells (see figure 4 in online supplement). The present findings did not depend on the kind of cancer cell transplanted.

To estimate the significance of preventing the diffusion of gaseous sulfur-containing compounds into the body, we performed a control experiment in which a mixture of D-methionine and L-lactic acid or D-glucose was injected subcutaneously into the right flank of nude mice and its effects on the cell tissue were observed after approximately 3 months. An inflammatory response was observed at the injection site and also around the inguinal lymph node and in the opposite left flank which had been regarded as healthy (see figure 5 in online supplement). For the inflamed tissues, immunohistochemistry detection revealed p53 gene products in the nuclei of the epithelial cells of the epidermis and of the hair follicles, which suggests the occurrence of mutation in the p53 gene (see figure 6 in online supplement). Since the p53 gene is very sensitive to genotoxic stress, we checked for anomalies in heme activity using anti Ki-67 rabbit polyclonal antibody (see figure 7 in online supplement).

We investigated how immunity deficiency, which is characteristic of nude mice, is related to tumour proliferation. Sarcoma-180 was transplanted to nude (T cell-deficient) and ICR mice (with T cells) at 2×106 per mouse and the tumour area over time relationship was estimated. The average proliferation rate for the ICR mice was approximately 47% lower than that for the nude mice. Moreover, spleen enlargement was more distinct for the ICR mice than for the nude mice (see figure 8 in online supplement).

It has been reported that hyaluronic acid (HA), which is a constituent of the extracellular matrix, breaks down into smaller molecules as tumour cells proliferate, thereby inducing angiogenesis of the tumour cells.29–31 To investigate how gaseous sulfur-containing compounds contribute to dissociation of Na-HA polymers, we compared the results when Na-HA solution was mixed with the plasma constituents of peripheral blood taken from lung cancer patients with those when Na-HA solution was mixed with plasma from which the gaseous sulfur-containing compounds had been removed (see Materials and methods section). The solutions produced under each condition were dripped onto a platinum grid, negatively stained using uranyl acetate solution, and then observed using TEM (figure 5). The TEM images for the control sample (original Na-HA solution) revealed fibrous structures in which the granular components were bound together (figure 5A). When the Na-HA was dissolved directly in plasma from a patient with lung cancer, the fibrous structures disappeared and the particles were separated (figure 5B). When it was dissolved in plasma that had been subjected to gas adsorption (figure 5C), the particles bonded together although a clear fibrous structure was not seen, indicating that the dissociation of Na-HA was suppressed. The repeated use of Na-HA therefore inhibits the diffusion of gaseous sulfur-containing compounds and thus tumour proliferation.

Figure 5

TEM photographs of sodium hyaluronate (Na-HA) solutions. (A) Control solution. Granular components bonded together and fibrous structures formed. (B) Solution mixed with plasma constituents of peripheral blood sampled from a patient with lung cancer showing the Na-HA polymer dissociated into small particles (arrows). (C) Solution mixed with plasma sample from a patient with lung cancer in which gaseous sulfur-containing compounds were removed by adsorption on rhodium nanoparticle-coated plate showing particles bonded together.

Discussion

This study showed that gaseous sulfur-containing compounds are generated in tumour tissues and that their diffusion into the surrounding tissues greatly affects tumour proliferation. There have been many reports on the generation of certain odorous compounds in tumour tissues, and attempts have been made to detect such substances using dogs to judge the presence or absence of cancers.32–34 Carolyn et al trained dogs to detect bladder cancer on the basis of urine odour. The success rate of the groups was 41%, and they concluded that the dogs distinguish the characteristic volatile compounds produced in bladder cancers.35 36 These results are consistent with ours, since the methanethiol and hydrogen sulfide detected in the flatus from the patients with colon cancer and the exhaled air from the patients with lung cancer are very volatile and are likely to be the main odorants generated in the cancer cells.

From our finding that sulfur-containing gas can be generated by reacting sulfur-containing amino acids with glucose or lactic acid, it can easily be presumed that accumulation of lactic acid (a metabolite of tumour cells) and glucose (required for tumour cell metabolism) results in the generation of sulfur-containing gas in tumour tissues. It is known that hydrogen sulfide gas binds strongly with the heme iron of methaemoglobin (MetHb) at the sixth ligand. Changes in the Raman spectrum confirmed that the S ions in the generated sulfur-containing gas were strongly bound to the heme iron of haemoglobin in the presence of glucose. Such binding of S ions with the heme iron of MetHb is considered to result from oxyhaemoglobin (OxyHb) being transformed into MetHb in the presence of glucose. It is known that hydrogen sulfide inhibits cytochrome C oxidase (COX).21

In tumour cells, mitochondrial respiration is impaired and changes occur in the subunit proteins. Matoba et al investigated the role of various genes in the structure and activity of the COX complex and found that oxygen consumption and COX enzyme activity were greatly reduced in the absence of the p53 gene.37 Furthermore, it has been reported that p53 is involved in the expression of synthesis of cytochrome C oxidase 2 (SCO2), an important factor in the regulation of modelling the mitochondria COX complex.38 39 Oxygen consumption in the mitochondria is reduced in p53-defective cells because SCO2 expression is low: this is compensated for by enhanced energy production via the glycolytic pathway.40 41 These results indicate that inhibition of COX by the sulfur-containing gas generated in tumour tissues affects p53 gene function39 which, in turn, reduces SCO2 expression.42

Anaerobic condition is created in the tumour tissues.43 It is well known that the cancer cells do not use the oxygen in their energetic metabolism even when there is enough oxygen in the surrounding environment.44 Our results suggest that sulfur-containing gases cause the respiratory depression on tissues and set them at histotoxic hypoxia.45

A possible approach to cancer therapy is therefore direct desulfurisation of the blood of cancer patients (as illustrated in figure 9 in the online supplement). Complete desulfurisation must of course be avoided because some of the essential amino acids in the blood contain sulfur. A method such as plasma purification could achieve desirable desulfurisation (blood normalisation) while affecting blood components as little as possible. It might be possible to develop oral drugs that eliminate sulfur-containing gas.

Acknowledgments

We thank Showa Yakuhin Co Ltd for providing the reagents and preparing the sodium hyaluronate solutions and Kyodo Byori Inc for helping with the tissue diagnosis.

References

Footnotes

  • Competing interests None.

  • Patient consent Obtained.

  • Ethics approval This study was conducted with the approval of Jichi-Ika University Hospital (Saitama, Japan) and Nagoya Central Hospital (Nagoya, Japan).

  • Provenance and peer review Not commissioned; externally peer reviewed.

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