The Role of CO₂ in Cancer Research and Care

Inhalation of 5% carbon dioxide (CO,) and 95% oxygen (0,)or 10% CO, and90% 0, at one a t m pressure increased blood flow of the C3HBA isotransplanta n d C3H isoimplant in C3H mice compared with animals breathing 100% 0,or air. Oxygen concentration, measured b y a platinum microelectrode, was im-proved in anoxic areas of the tumor when the animals inhaled the gases con-taining CO, compared with those breathing 100% 0, or air; the relative increasewas less when the electrode was placed in a well-vascularized area. Rectal tem-perature, heart a n d respiration rates were decreased in animals breathing thegases containing CO, compared with air; rectal temperature was increased b yinhalation of 100% 0,. The results suggest that inhalation of a gas mixturecontaining CO, a t atmospheric pressure during radiotherapy or chemotherapymay be of value i n treating cancer in humans

Mitochondria play an essential role in cellular energy metabolism and apoptosis. Previous studies have demonstrated that decreased mitochondrial biogenesis is associated with cancer progression. In mitochondrial biogenesis, peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) regulates the activities of multiple nuclear receptors and transcription factors involved in mitochondrial proliferation. Previously, we showed that overexpression of PGC-1α leads to mitochondrial proliferation and induces apoptosis in human malignant fibrous histiocytoma (MFH) cells in vitro. We also demonstrated that transcutaneous application of carbon dioxide (CO(2)) to rat skeletal muscle induces PGC-1α expression and causes an increase in mitochondrial proliferation. In this study, we utilized a murine model of human MFH to determine the effect of transcutaneous CO(2)) exposure on PGC-1α expression, mitochondrial proliferation and cellular apoptosis. PGC-1α expression was evaluated by quantitative real-time PCR, while mitochondrial proliferation was assessed by immunofluorescence staining and the relative copy number of mitochondrial DNA (mtDNA) was assessed by real-time PCR. Immunofluorescence staining and DNA fragmentation assays were used to examine mitochondrial apoptosis. We also evaluated the expression of mitochondrial apoptosis related proteins, such as caspases, cytochorome c and Bax, by immunoblot analysis. We show that transcutaneous application of CO(2)) induces PGC-1α expression, and increases mitochondrial proliferation and apoptosis of tumor cells, significantly reducing tumor volume. Proteins involved in the mitochondrial apoptotic cascade, including caspase 3 and caspase 9, were elevated in CO(2)) treated tumors compared to control. We also observed an enrichment of cytochrome c in the cytoplasmic fraction and Bax protein in the mitochondrial fraction of CO(2)) treated tumors, highlighting the involvement of mitochondria in apoptosis. These data indicate that transcutaneous application of CO(2)) may represent a novel therapeutic tool in the treatment of human MFH.

Hypoxia plays a significant role in cancer progression, including metastatic bone tumors. We previously reported that transcutaneous carbon dioxide (CO2) application could decrease tumor progression through the improvement of intratumor hypoxia. Therefore, we hypothesized that decreased hypoxia using transcutaneous CO2 could suppress progressive bone destruction in cancer metastasis. In the present study, we examined the effects of transcutaneous CO2 application on metastatic bone destruction using an animal model. The human breast cancer cell line MDA-MB-231 was cultured in vitro under three different oxygen conditions, and the effect of altered oxygen conditions on the expression of osteoclast-differentiation and osteolytic factors was assessed. An in vivo bone metastatic model of human breast cancer was created by intramedullary implantation of MDA-MB-231 cells into the tibia of nude mice, and treatment with 100% CO2 or a control was performed twice weekly for two weeks. Bone volume of the treated tibia was evaluated by micro-computed tomography (µCT), and following treatment, histological evaluation was performed by hematoxylin and eosin staining and immunohistochemical staining for hypoxia-inducible factor (HIF)-1α, osteoclast-differentiation and osteolytic factors, and tartrate-resistant acid phosphatase (TRAP) staining for osteoclast activity. In vitro experiments revealed that the mRNA expression of RANKL, PTHrP and IL-8 was significantly increased under hypoxic conditions and was subsequently reduced by reoxygenation. In vivo results by µCT revealed that bone destruction was suppressed by transcutaneous CO2, and that the expression of osteoclast-differentiation and osteolytic factors, as well as HIF-1α, was decreased in CO2-treated tumor tissues. In addition, multinucleated TRAP-positive osteoclasts were significantly decreased in CO2-treated tumor tissues. Hypoxic conditions promoted bone destruction in breast cancer metastasis, and reversal of hypoxia by transcutaneous CO2 application significantly inhibited metastatic bone destruction along with decreased osteoclast activity. The findings in this study strongly indicated that transcutaneous CO2 application could be a novel therapeutic strategy for treating metastatic bone destruction.

Carbon dioxide (CO2) therapy can be applied to treat a variety of disorders. We previously found that transcutaneous application of CO2 with a hydrogel decreased the tumor volume of several types of tumors and induced apoptosis via the mitochondrial pathway. However, only one condition of treatment intensity has been tested. For widespread application in clinical antitumor therapy, the conditions must be optimized. In the present study, we investigated the relationship between the duration, frequency, and treatment interval of transcutaneous CO2 application and antitumor effects in murine xenograft models. Murine xenograft models of three types of human tumors (breast cancer, osteosarcoma, and malignant fibrous histiocytoma/undifferentiated pleomorphic sarcoma) were used to assess the antitumor effects of transcutaneous CO2 application of varying durations, frequencies, and treatment intervals. In all human tumor xenografts, apoptosis was significantly induced by CO2 treatment for ≥10 min, and a significant decrease in tumor volume was observed with CO2 treatments of >5 min. The effect on tumor volume was not dependent on the frequency of CO2 application, i.e., twice or five times per week. However, treatment using 3- and 4-day intervals was more effective at decreasing tumor volume than treatment using 2- and 5-day intervals. The optimal conditions of transcutaneous CO2 application to obtain the best antitumor effect in various tumors were as follows: greater than 10 min per application, twice per week, with 3- and 4-day intervals, and application to the site of the tumor. The results suggest that this novel transcutaneous CO2 application might be useful to treat primary tumors, while mitigating some side effects, and therefore could be safe for clinical trials.

Squamous cell carcinoma (SCC) is the main histological type of oral cancer. Its growth rate and incidence of metastasis to regional lymph nodes is influenced by various factors, including hypoxic conditions. We have previously reported that transcutaneous CO2 induces mitochondrial apoptosis and decreases lung metastasis by reoxygenating sarcoma cells. However, previous studies have not determined the sequential mechanism by which transcutaneous CO2 suppresses growth of epithelial tumors, including SCCs. Moreover, there is no report that transcutaneous CO2 suppresses lymphogenous metastasis using human cell lines xenografts. In this study, we examined the effects of transcutaneous CO2 on cancer apoptosis and lymphogenous metastasis using human SCC xenografts. Our results showed that transcutaneous CO2 affects expressions of PGC-1α and TFAM and protein levels of cleavage products of caspase-3, caspase-9 and PARP, which relatives mitochondrial apoptosis. They also showed that transcutaneous CO2 significantly inhibits SCC tumor growth and affects expressions of HIF-1α, VEGF, MMP-2 and MMP-9, which play essential roles in tumor angiogenesis, invasion and metastasis. In conclusion, transcutaneous CO2 suppressed tumor growth, increased mitochondrial apoptosis and decreased the number of lymph node metastasis in human SCC by decreasing intra-tumoral hypoxia and suppressing metastatic potential with no observable effect in vivo. Our findings indicate that transcutaneous CO2 could be a novel therapeutic tool for treating human SCC.

Hypoxia has been associated with poor local tumour control and relapse in many cancer sites, including carcinoma of the prostate. This translational study tests whether breathing carbogen gas improves the oxygenation of human prostate carcinoma xenografts in mice and in human patients with prostate cancer. A total of 23 DU145 tumour-bearing mice, 17 PC3 tumour-bearing mice and 17 human patients with prostate cancer were investigated. Intrinsic susceptibility-weighted MRI was performed before and during a period of carbogen gas breathing. Quantitative R(2)* pixel maps were produced for each tumour and at each time point and changes in R(2)* induced by carbogen were determined. There was a mean reduction in R(2)* of 6.4% (P=0.003) for DU145 xenografts and 5.8% (P=0.007) for PC3 xenografts. In all, 14 human subjects were evaluable; 64% had reductions in tumour R(2)* during carbogen inhalation with a mean reduction of 21.6% (P=0.0005). Decreases in prostate tumour R(2)* in both animal models and human patients as a result of carbogen inhalation suggests the presence of significant hypoxia. The finding that carbogen gas breathing improves prostate tumour oxygenation provides a rationale for testing the radiosensitising effects of combining carbogen gas breathing with radiotherapy in prostate cancer patients.

Noninvasive and targeted physical treatment is still desirable especially for those cancerous patients. Herein, we develop a new physical treatment protocol by employing CO2 bubbling-based 'nanobomb' system consisting of low-intensity ultrasound (1.0 W/cm(2)) and a well-constructed pH/temperature dual-responsive CO2 release system. Depending on the temperature elevation caused by exogenous low-intensity therapeutic ultrasound irradiation and the low pH caused by the endogenous acidic-environment around/within tumor, dual-responsive CO2 release system can quickly release CO2 bubbles, and afterwards, the generated CO2 bubbles waves will timely explode before dissolution due to triggering by therapeutic ultrasound waves. Related bio-effects (e.g., cavitation, mechanical, shock waves, etc) caused by CO2 bubbles' explosion effectively induce instant necrosis of panc-1 cells and blood vessel destruction within panc-1 tumor, and consequently inhibit the growth of panc-1 solid tumor, simultaneously minimizing the side effects to normal organs. This new physiotherapy employing CO2 bubbling-based 'nanobomb' system promises significant potentials in targetedly suppressing tumors, especially for those highly deadly cancers. Keywords: Nanobomb; dual-responsive CO2 release; inertial cavitation; low intensity ultrasound; panc-1 pancreatic cancer.

The present study aimed to evaluate the efficacy of an intra-arterially infused carbon dioxide (CO2)-saturated solution in sensitizing the anticancer effect of cisplatin in a rabbit VX2 liver tumor model. Forty VX2 liver tumor-bearing Japanese white rabbits were randomly divided into four groups and infused via the proper hepatic artery with a saline solution (control group), CO2-saturated solution (CO2 group), cisplatin solution (cisplatin group), or CO2-saturated solution and cisplatin solution (combined group). The tumor volume (TV) and the relative tumor volume (RTV), RTV = (TV on day 3 or 7)/(TV on day 0) x 100, were calculated using contrast-enhanced computed tomography. Hypoxia-inducible factor-1α (HIF‑1α) and carbonic anhydrase IX (CA IX) staining were used to evaluate cellular hypoxia. Cleaved caspase-3 and cleaved caspase-9 were analyzed to assess tumor apoptosis. The mean RTV on days 3 and 7 were 202.6±23.7 and 429.2±94.8%, respectively, in the control group; 172.2±38.1 and 376.5±61.1% in the CO2 group; 156.1±15.1 and 269.6±45.2% in the cisplatin group; and 118.3±28.1 and 210.3±55.1% in the combined group. RTV was significantly lower in the CO2 group than in the control group (day 3; P<0.05), and in the combined group than in the cisplatin group (days 3 and 7; P<0.05). HIF-1α and CA IX suppression, and increased cleaved caspase-3 and cleaved caspase-9 expression, were detected in the CO2 and combined groups, compared with the other two groups. An intra-arterially infused CO2-saturated solution inhibits liver VX2 tumor growth and sensitizes the anticancer effect of cisplatin.

Introduction: In recent years, the tumour immunosuppressive mechanism has attracted attention as a cause of tumour chemoresistance. Although chemoresistance and immunosuppression of tumours have been reported to be associated with a hypoxic environment, effective treatments to improve hypoxia in tumours have not yet been established. We have previously applied carbon dioxide (CO2) to squamous cell carcinoma and have shown that improvement in local oxygenation has an antitumour effect. However, the effects of local CO2 administration on tumour immunosuppression, chemoresistance, and combination with chemotherapy are unknown. In this study, we investigated the effects of local CO2 administration on squamous cell carcinoma and the effects of combined use with chemotherapy, focusing on the effects on tumour immunosuppressive factors. Methods: Human oral squamous cell carcinoma (HSC-3) was transplanted subcutaneously into the back of a nude mouse, and CO2 and cisplatin were administered. After administration twice a week for a total of 4 times, tumours were collected and the expression of tumour immunosuppressive factors (PD-L1, PD-L2, and galectin-9) was evaluated using real-time polymerase chain reaction and immunostaining. Results: Compared with the control group, a significant decrease in the mRNA expression of PD-L1 was observed in both, CO2-treated and combination groups. Similarly, the expression of PD-L2 and galectin-9 decreased in the CO2-treated and combination groups. Furthermore, immunostaining also showed a significant decrease in the protein expression of tumour immunosuppressive factors in the CO2-treated and combination groups. Conclusion: It was confirmed that the tumour immunosuppressive factors decreased due to local CO2 administration to the mouse model. CO2 administration has the potential to improve the hypoxic environment in tumours, and combined use with chemotherapy may also improve tumour immunosuppression.

Osteosarcoma is the most common primary solid malignant bone tumor. Despite substantial improvements in surgery and chemotherapy, metastasis remains a major cause of fatal outcomes, and the molecular mechanisms of metastasis are still poorly understood. Hypoxia, which is common in malignant tumors including osteosarcoma, increases expressions of hypoxia inducible factor (HIF)-1α, matrix metalloproteinase (MMP)-2 and MMP-9, and can induce invasiveness. As we previously showed a novel transcutaneous CO2 application to decrease HIF-1α expression and induce apoptosis in malignant fibrous histiocytoma, we hypothesize that transcutaneous CO2 application could suppress metastatic potential of osteosarcoma by improving hypoxic conditions. Here, we examined the effects of transcutaneous CO2 application on apoptosis, and development of pulmonary metastasis using a highly metastatic osteosarcoma cell line, LM8. Transcutaneous CO2 application significantly decreased tumor growth and pulmonary metastasis in LM8 cells. Apoptotic activity increased, and intratumoral hypoxia was improved with decreased expressions of HIF-1α, MMP-2 and MMP-9, significantly, in the CO2-treated tumors. In conclusion, we found that transcutaneous CO2 application can induce tumor cell apoptosis and might suppress pulmonary metastasis by improvement of hypoxic conditions with decreased expressions of HIF-1α and MMPs in highly metastatic osteosarcoma cell. These findings strongly indicate that this novel transcutaneous CO2 therapy could be a therapeutic breakthrough for osteosarcoma patients.

Keywords: Hypoxia; Matrix metalloproteinase (MMP); Metastasis; Osteosarcoma; Transcutaneous CO(2) application.

Cancer cachexia causes skeletal muscle atrophy, impacting the treatment and prognosis of patients with advanced cancer, but no treatment has yet been established to control cancer cachexia. We demonstrated that transcutaneous application of carbon dioxide (CO2) could improve local blood flow and reduce skeletal muscle atrophy in a fracture model. However, the effects of transcutaneous application of CO2 in cancer-bearing conditions are not yet known. In this study, we calculated fat-free body mass (FFM), defined as the skeletal muscle mass, and evaluated the expression of muscle atrophy markers and uncoupling protein markers as well as the cross-sectional area (CSA) to investigate whether transcutaneous application of CO2 to skeletal muscle could suppress skeletal muscle atrophy in cancer-bearing mice. Human oral squamous cell carcinoma was transplanted subcutaneously into the upper dorsal region of nude mice, and 1 week later, CO2 gas was applied to the legs twice a week for 4 weeks and FFM was calculated by bioimpedance spectroscopy. After the experiment concluded, the quadriceps were extracted, and muscle atrophy markers (muscle atrophy F-box protein (MAFbx), muscle RING-finger protein 1 (MuRF-1)) and uncoupling protein markers (uncoupling protein 2 (UCP2) and uncoupling protein 3 (UCP3)) were evaluated by real-time polymerase chain reaction and immunohistochemical staining, and CSA by hematoxylin and eosin staining. The CO2-treated group exhibited significant mRNA and protein expression inhibition of the four markers. Furthermore, immunohistochemical staining showed decreased MAFbx, MuRF-1, UCP2, and UCP3 in the CO2-treated group. In fact, the CSA in hematoxylin and eosin staining and the FFM revealed significant suppression of skeletal muscle atrophy in the CO2-treated group. We suggest that transcutaneous application of CO2 to skeletal muscle suppresses skeletal muscle atrophy in a mouse model of oral squamous cell carcinoma.

Sarcomas are relatively resistant because of hypoxia. We previously demonstrated that the transcutaneous CO(2) therapy reduced hypoxic conditions in human malignant fibrous histiocytoma (MFH). Therefore, we hypothesized that transcutaneous CO(2) therapy could enhance the antitumor effect of radiation therapy in human MFH. Our purpose was to evaluate the effects of transcutaneous CO(2) therapy on the antitumor efficacy of X-ray irradiation using MFH. First, in an in vitro study, we assessed apoptotic activity and reactive oxygen species (ROS) production using flow cytometric and immunoblot analysis at 24 h after X-ray irradiation under three different oxygen conditions (normoxic, reoxygenated and hypoxic). In addition, in the in vivo study, 24 male athymic BALB/c nude mice with MFH tumors that were inoculated in the dorsal subcutaneous area were randomized into four groups: control, CO(2), X-ray irradiation and combination (CO(2) and X-ray irradiation). Treatments were performed twice weekly for 2 weeks, four times in total. Tumor volume was calculated. All tumors were excised and apoptotic activity, ROS production, related proteins and HIF-1α expression were assessed using flow cytometric and immunoblot analysis. The in vitro study revealed that X-ray irradiation induced increased apoptosis and ROS production in MFH cells under normoxic and reoxygenated conditions relative to hypoxic conditions (P<0.01). In the in vivo study, tumor volume in the combination group was reduced to 28, 42 and 47% of that in the control, CO(2), and X-ray groups, respectively (P<0.05). Apoptotic activity and ROS production in the combination group were strongly increased with decreasing HIF-1α expression relative to the control, CO(2) and X-ray groups. The transcutaneous CO(2) system enhanced the antitumor action of X-ray irradiation and could be a novel therapeutic tool for overcoming radio-resistance in human malignancies.

Background: Tumour hypoxia limits the effectiveness of radiation therapy. Delivering normobaric or hyperbaric oxygen therapy elevates pO2 in both tumour and normal brain tissue. However, pO2 levels return to baseline within 15 minutes of stopping therapy.

Aim: To investigate the effect of perfluorocarbon (PFC) emulsions on hypoxia in subcutaneous and intracranial mouse gliomas and their radiosensitising effect in orthotopic gliomas in mice breathing carbogen (95%O2 and 5%CO2).

Results: PFC emulsions completely abrogated hypoxia in both subcutaneous and intracranial GL261 models and conferred a significant survival advantage orthotopically (Mantel Cox: p = 0.048) in carbogen breathing mice injected intravenously (IV) with PFC emulsions before radiation versus mice receiving radiation alone. Carbogen alone decreased hypoxia levels substantially and conferred a smaller but not statistically significant survival advantage over and above radiation alone.

Conclusion: IV injections of PFC emulsions followed by 1h carbogen breathing, radiosensitises GL261 intracranial tumors.

Gas-involving cancer theranostics have attracted considerable attention in recent years due to their high therapeutic efficacy and biosafety. We have reviewed the recent significant advances in the development of stimuli-responsive gas releasing molecules (GRMs) and gas nanogenerators for cancer bioimaging, targeted and controlled gas therapy, and gas-sensitized synergistic therapy. We have focused on gases with known anticancer effects, such as oxygen (O2), carbon monoxide (CO), nitric oxide (NO), hydrogen sulfide (H2S), hydrogen (H2), sulfur dioxide (SO2), carbon dioxide (CO2), and heavy gases that act via the gas-generating process. The GRMs and gas nanogenerators for each gas have been described in terms of the stimulation method, followed by their applications in ultrasound and multimodal imaging, and finally their primary and synergistic actions with other cancer therapeutic modalities. The current challenges and future possibilities of gas therapy and imaging vis-à-vis clinical translation have also been discussed.

Keywords: cancer imaging; cancer therapy; controlled release; endogenous/exogenous stimulation; gas nanogenerators; gas releasing system; gas therapy; synergetic therapy; theranostic.

Gas therapy appears promising in treating cancer, ischemia-reperfusion injury, heart attack, and acute situations of shock. Gas therapy is also becoming a good choice in preventive medicine against dementia, reducing cancer occurrence, cardiovascular diseases, and delaying aging. On-demand gas release allows precision therapy with few risks of gas poisoning. Here, we review targeted and controlled gas therapy, gas nanogenerators for cancer bioimaging, and gas-sensitized synergistic therapy. We focus on gases with anticancer effects such as hydrogen, carbon monoxide, carbon dioxide, nitric oxide, oxygen sulfur dioxide, hydrogen sulfide, and heavy gases. We discuss the efficiency of gas therapy combined with other therapies methods such as starvation therapy, ultrasound therapy, chemotherapy, radiotherapy, and photodynamic therapy, and applications in multimodal and ultrasound imaging.

Background: Laparoscopic CO2-insufflation is believed to stimulate proliferation and metastatic potential of gastrointestinal carcinomas. E-cadherin, I-CAM1, I-CAM2, and CD44 are involved into the metastatic process of different cancer cell lines. The current study investigates the influence of CO2-insufflation on the expression of E-cadherin, I-CAM1, I-CAM2, and CD44 in vitro.

Methods: CX-2 and CC531 colon carcinoma cells (human/rat) were exposed to pneumoperitoneal CO2-insufflation. E-cadherin, I-CAM1, I-CAM2, and CD44 were measured 0, 12, 24, 48, and 72 h after CO2-insufflation using flowcytometry. Control groups were exposed to room air. Data were analyzed by the Wilcoxon-Mann-Whitney U-test.

Results: Both cell lines showed significant alteration in E-cadherin, I-CAM1, and CD44 expression after CO2 exposure (p <0.05). No significant differences were found regarding I-CAM2 expression.

Conclusion: The present study demonstrates CO2-insufflation to influence the expression of E-cadherin, I-CAM1 and CD44. Whether these changes increases the metastatic potential of colorectal cancer cells in vivo needs further investigation.

Purpose: To increase pulmonary deposition of anticancer liposome aerosols in mice by modulation of respiratory physiology through the addition of 5% CO2 to the air source used to generate the aerosols. Breathing CO2-enriched aerosol increases pulmonary ventilation with concurrent increased deposition of inhaled particles.

Methods: Dilauroylphosphatidylcholine liposome formulations of two anticancer drugs, paclitaxel (PTX) and camptothecin (CPT), were investigated. The aerosol droplet size was measured using an Andersen cascade impactor. Drug concentrations in aerosol droplet fractions and tissues were determined by HPLC analysis. ICR mice were exposed to each liposome aerosol for 30 min. For each drug, one group of mice inhaled the drug-liposome aerosol generated with a mixture of 5% CO2 in air and another group inhaled the drug-liposome aerosols produced with normal air. Tissue distribution and pharmacokinetics were determined for both drug delivery systems.

Results: Significantly higher concentrations of PTX and CPT were found in organs of mice exposed to 5% CO2-air aerosols compared to organs of mice exposed to normal air aerosols. The highest concentrations of drug were detected in the lungs and were two- to fourfold higher with 5% CO2-air aerosols than with aerosols generated with normal air. Higher concentrations were also detected in liver, spleen, kidneys, blood, and brain.

Conclusion: 5% CO2 enrichment of air increased respiratory tract deposition of inhaled aerosol particles containing PTX and CPT.

Background: CO2 pneumoperitoneum provides a new surgical environment to treat malignant disease. The purpose of this study was to investigate the influence of CO2 pneumoperitoneum during laparoscopic surgery on cancer cell growth.

Methods: WiDr human colon cancer cells were incubated for 3 h under the following two conditions: 100% CO2 at 10 mmHg, and 95% air/5% CO2 (control). Cell proliferation was assessed by the WST-1 assay and BrdU assay. Tumor growth was assessed by subcutaneous injection into 20 nude mice. Cellular damage was measured by lactate dehydrogenase (LDH) assay.

Results: The number of WiDr cells under pneumoperitoneal conditions decreased in the first 24 h. However, no significant difference was observed in the proliferation rate and tumor growth of the viable cells. LDH release of the CO2 pneumoperitoneal group was higher than that of the controls.

Conclusions: Our data indicate that CO2 pneumoperitoneum does not promote cancer cell proliferation but instead has a toxic effect on cancer cells.

In order to meet the high energy demand, a metabolic reprogramming occurs in cancer cells. Its role is crucial in promoting tumor survival. Among the substrates in demand, oxygen is fundamental for bioenergetics. Nevertheless, tumor microenvironment is frequently characterized by low-oxygen conditions. Hypoxia-inducible factor 1 (HIF-1) is a pivotal modulator of the metabolic reprogramming which takes place in hypoxic cancer cells. In the hub of cellular bioenergetics, mitochondria are key players in regulating cellular energy. Therefore, a close crosstalk between mitochondria and HIF-1 underlies the metabolic and functional changes of cancer cells. Noteworthy, HIF-1 represents a promising target for novel cancer therapeutics. In this review, we summarize the molecular mechanisms underlying the interplay between HIF-1 and energetic metabolism, with a focus on mitochondria, of hypoxic cancer cells.

Keywords: HIF-1; TCA cycle; cancer metabolism; hypoxia; mitochondria.

We previously performed MRI studies of HCC (hepatocellular carcinomas) in mice showing the feasibility of measuring a carbogen effect. In the present study carbogen response of the whole tumour was compared with growth characteristics during longitudinal follow-up. HCC were chemically induced. The imaging protocol at 4.7 T comprised a fast spin-echo sequence for high-resolution screening and measurement of growth curves, and a fast gradient echo sequence allowing an entire T2*w image acquisition per respiratory cycle to perform fMRI under carbogen breathing. A new parameter, T+, the fraction of tumour voxels with increased intensity under carbogen was measured on manually defined ROIs. Twenty-two HCC were followed for 3-10 weeks. Tumours were divided into two groups, "regularly" and "irregularly" growing tumours. A linear correlation between T+ and tumour growth rate was observed only for "regularly" growing HCC. These results suggest a link between tumour growth rates and tumour fractions exhibiting signal increase upon carbogen breathing. They are compatible with observations by others that rapidly growing tumours are more hypoxic than slowly growing ones. Combined measurement of T+ and tumour growth may become a useful noninvasive follow-up approach for assessment and/or management of therapies involving vasculature-targeting and anti-proliferative drugs.

The aim of this study was to evaluate the effect of carbogenbreathing on the physiological profile of human glioma xeno-grafts. Near infrared spectroscopy was used to investigatechanges in oxy- and deoxyhemoglobin concentrations in tumorblood. Oxygen tension changes in tumor tissue were evaluatedby 19F-MR relaxometry, using perfluoro-15-crown-5-ether, andmodifications of tumor blood perfusion (TBP) were analyzed byfast dynamic 1H-MR imaging of Gd-DTPA uptake. Finally, changesof the bioenergetic status and pH of tumor cells were analyzedby 31P-MRS. After 5 to 8 min of carbogen breathing, the averageoxygen tension increase in tumor tissue was 4.6 6 1.3 mm Hg,which is in agreement with an increase of the oxyhemoglobinconcentration in tumor blood (D[O 2Hb] 5 9.2 6 3 mM). However,simultaneously the TBP was reduced, the bioenergetic statuswas diminished, and pH was decreased. As 100% O 2 breathingalone did not result in a detectable increase of oxyhemoglobinin tumor blood, the increase of the tumor oxygenation bycarbogen appears to be mediated by its CO 2 content. Thiscomponent may cause a nutrient-limited decrease of oxidativeenergy metabolism, indirectly via a steal-effect and/or by inhibi-tion of the glycolytic rate resulting from tissue acidification.Magn Reson Med 42:490–499, 1999. r 1999 Wiley-Liss, Inc.Key words: gliomas; carbogen; perfusion; oxygenation; bioen-ergetic status

Background: This study aimed to investigate the growth curve, cell colony formation, cell cycle, apoptosis, anti-anoikis, and ability of invasion, adhesion, and migration of cervical cancer cells after exposure to a model of a simulated CO2 pneumoperitoneum environment with different pressures and at different times.

Material and methods: The cervical cancer cells were cultured in groups with 8 and 16 mmHg of 100% CO2 for 1, 2, 3, and 4 h in a model of a simulated environment of CO2 pneumoperitoneum. The cells in the control group were cultured in a standard environment. The growth curve was drawn through constant survival cell counts for 7 days, and the group with most obvious change was selected for subsequent experiments to detect cell colony formation, cell cycle apoptosis, and anti-anoikis, and the ability of invasion, adhesion, and migration.

Results: After a brief inhibition, the proliferation of cervical cancer cells was markedly increased and had no relationship with different CO2 pressures. Compared with the control group, the early apoptosis rate in the experimental group was higher, and the ability of invasion, migration, and adhesion decreased significantly.

Conclusions: Cervical cancer cells stimulated by a CO2 pneumoperitoneum environment in vitro have an increased the ability to proliferate after a short period of inhibition and have reduced abilities of invasion, migration, and adhesion.

The effect of Fluosol DA 20%, an emulsion of perfluorochemicals, in combination with carbogen (95% O2 and 5% CO2) breathing on the response of mouse tumors to radiation was studied. When A/J mice bearing SCK tumors in the right hind limb were injected iv with Fluosol DA 20% at 12 ml/kg and exposed to carbogen for 1 h before and during the irradiation of tumors, the response of tumors to a single dose of X irradiation was significantly enhanced. The dose modification factors for growth delay and cure of SCK tumors were 2.10 +/- 0.01 (SE) and 1.86 +/- 0.18 (SE), respectively. Such a treatment slightly increased the radiation-induced skin damage by a factor of 1.17 +/- 0.02 (SE), resulting in a therapeutic gain of 1.79 +/- 0.01 (SE) for the growth delay and 1.59 +/- 0.09 (SE) for the curability. Carbogen breathing alone also increased the response of tumor and skin to radiation, but it was far less effective than the combination of Fluosol DA 20% and carbogen breathing. It was concluded that iv injection of Fluosol DA 20% in conjunction with carbogen breathing significantly increased the O2 transport to hypoxic areas in the SCK tumors and thus significantly enhanced the tumoricidal effect of radiation on SCK tumors.

Background: Peritoneal carcinomatosis (PC) arising from colorectal cancer is associated with poor prognosis and few treatment options are currently available. Laparoscopic CO2 insufflation stimulates the progression and metastatic potential of gastrointestinal carcinomas. However, heated and humidified CO2 pneumoperitoneum (HH-CO2) is a promising treatment for PC, although its effects and mechanism of action in human colon cancer cells remain unclear. This study evaluated the anti-tumour effects of HH-CO2 on human colon cancer in vitro.

Methods: Cell viability was assessed using the WST-8 assay in two colon cancer cell lines. Apoptosis was assessed by Annexin V PI flow cytometry, and migration and invasion were examined using wound healing and Transwell® invasion assays. The expressions of Bcl-2, Bax, matrix metalloproteinase-2 (MMP-2), E-cadherin, ICAM-1, and CD44 were detected by western blotting.

Results: HH-CO2 significantly inhibited cell proliferation, migration, invasion and adhesion. HH-CO2 induced apoptosis and significantly inhibited the expression of Bcl-2, MMP-2, ICAM-1 and CD44, and increased Bax and E-cadherin expression in colon cancer cells.

Conclusions: HH-CO2 induces apoptosis and inhibits proliferation, migration, invasion and adhesion of human colon cancer cells. Our results suggest that HH-CO2 may serve as a potential candidate for the treatment and/or prevention of peritoneal carcinomatosis from colorectal cancer and warrant further in vivo investigation.

Having a hypoxic microenvironment is a common and salient feature of most solid tumors. Hypoxia has a profound effect on the biological behavior and malignant phenotype of cancer cells, mediates the effects of cancer chemotherapy, radiotherapy, and immunotherapy through complex mechanisms, and is closely associated with poor prognosis in various cancer patients. Accumulating studies have demonstrated that through normalization of the tumor vasculature, nanoparticle carriers and biocarriers can effectively increase the oxygen concentration in the tumor microenvironment, improve drug delivery and the efficacy of radiotherapy. They also increase infiltration of innate and adaptive anti-tumor immune cells to enhance the efficacy of immunotherapy. Furthermore, drugs targeting key genes associated with hypoxia, including hypoxia tracers, hypoxia-activated prodrugs, and drugs targeting hypoxia-inducible factors and downstream targets, can be used for visualization and quantitative analysis of tumor hypoxia and antitumor activity. However, the relationship between hypoxia and cancer is an area of research that requires further exploration. Here, we investigated the potential factors in the development of hypoxia in cancer, changes in signaling pathways that occur in cancer cells to adapt to hypoxic environments, the mechanisms of hypoxia-induced cancer immune tolerance, chemotherapeutic tolerance, and enhanced radiation tolerance, as well as the insights and applications of hypoxia in cancer therapy.

Background This paper reports a study of 21 patients with peritoneal carcinomatosis from ovarian cancer who underwent cytoreductive surgery and HIPEC by means of PRS-1.0 Combat®, a new model for closed abdomen HIPEC aimed at improving fluid distribution with assistance from a CO2 recirculation system. This new technology has been previously shown to be successful in an experimental study (pig model) performed by our group, and has been approved for use in our hospital. Methods Twenty-one patients with peritoneal carcinomatosis of ovarian cancer origin were included in the study. Cytoreductive surgery and HIPEC were performed by a closed abdomen fluid and CO2 recirculation technique using the PRS-1.0 Combat(®) model. We analysed the intraoperative safety tolerance and post-operative morbidity and mortality during the first 30 days. Results Between November 2011 and March 2014 21 patients with epithelial ovarian cancer, International Federation of Gynecology and Obstetrics stage II-IV, were included in the study. During the procedure there were no significant haemodynamic or analytical disturbances. Complication rates were 38.1% and 57.14% for grade III/IV and minor (grade I/II) complications, respectively. Post-operative mortality was 4.76% (one patient). Complete cytoreductive surgery and intraperitoneal chemotherapy improved overall survival and disease-free survival in women with advanced ovarian cancer. The association of intra-abdominal hyperthermia with chemotherapy (HIPEC) increased the therapeutic benefit. Conclusions This study has shown that closed abdomen intraperitoneal chemohyperthermia by a fluid and CO2 recirculation system (PRS-1.0 Combat(®)) can be a safe and feasible model for the treatment of peritoneal carcinomatosis of ovarian cancer origin.

Keywords: Ovarian cancer; closed abdomen HIPEC.

Hypoxia and angiogenesis can significantly influence the efficacy of cancer therapy and the behavior of surviving tumor cells. There is a growing demand for technologies to measure tumor hypoxia and angiogenesis temporally in vivo to enable advances in drug development and optimization. This paper reports the use of frequency-domain photon migration with a side-firing probe to quantify tumor oxygenation and hemoglobin concentrations in nude rats bearing human head/neck tumors administered with carbogen gas, cycling hypoxic gas or just room air. Significant increase (with carbogen gas breathing) or decrease (with hypoxic gas breathing) in tumor oxygenation was observed. The trend in tumor oxygenation during forced cycling hypoxia (CH) followed that of the blood oxygenation measured with a pulse oximeter. Natural CH was also observed in rats under room air. The studies demonstrated the potential of the technology for longitudinal monitoring of tumor CH during tumor growth or in response to therapy.

Keywords: Diffuse optical spectroscopy; cancer therapy; fiber optic sensor; tumor hypoxia and angiogenesis.

Reactive oxygen species (ROS), a class of highly bioactive molecules, have been widely studied in various types of cancers. ROS are considered to be normal byproducts of numerous cellular processes. Typically, cancer cells exhibit higher basal levels of ROS compared with normal cells as a result of an imbalance between oxidants and antioxidants. ROS have a dual role in cell metabolism: At low to moderate levels, ROS act as signal transducers to activate cell proliferation, migration, invasion, and angiogenesis. In contrast, high levels of ROS cause damage to proteins, nucleic acids, lipids, membranes, and organelles, leading to cell death. Extensive studies have revealed that anticancer therapies that manipulate ROS levels, including immunotherapies, show promising in vitro as well as in vivo results. In this review, we summarize molecular mechanisms and oncogenic functions that modulate ROS levels and are useful for the development of cancer therapeutic strategies. This review also provides insights into the future development of effective agents that regulate the redox system for cancer treatment.

Keywords: cell death; neoplasms; oxidative stress; reactive oxygen species; therapeutics.

Hyperbaric oxygen (HBO) has been proposed to reduce tumour hypoxia by increasing the amount of dissolved oxygen in the plasma. That this actually occurs has not been verified experimentally. This study was performed to explore changes in tumour oxygenation induced by treatment with normobaric and hyperbaric oxygen and carbogen. R3230Ac mammary adenocarcinomas were implanted into Fisher 344 rats. Arterial blood gases, blood pressure and heart rate were monitored. Tumour oxygenation was measured polarographically in five sets of animals. They received either normobaric 100% oxygen, hyperbaric (3 atmospheres; atm) 100% oxygen, normobaric carbogen or hyperbaric (3 atm) carbogen (HBC) +/- bretylium. HBO reduced the mean level of low pO2 values (< 5 mmHg) from 0.49 to 0.07 (P = 0.0003) and increased the average median pO2 from 8 mmHg to 55 mmHg (P = 0.001). HBC reduced the level of low pO2 values from 0.82 to 0.51 (P = 0.002) an increased median pO2 from 2 mmHg to 6 mmHg (P = 0.05). Normobaric oxygen and carbogen did not change tumour oxygenation significantly. Sympathetic blockade with bretylium before HBC exposure improved oxygenation significantly more than HBC alone (low pO2 0.55-0.17, median pO2 4-17 mmHg). HBO and hyperbaric carbogen improved tumour oxygenation in this model, while normobaric oxygen or carbogen had no effect. Sympathetic-mediated vasoconstriction during hyperbaric carbogen caused it to be less effective than HBO. This mechanism also appeared to operate during normobaric carbogen breathing.