**4.1. Apoptotic cell killing**

38 Hyperthermia

It has been long known that hyperthermia can cause the softening or melting of the lipid bilayer [307], [308], [155], it can change lipid-protein interactions [309], and it can denature

Heat-treatment cause structural alteration in transmembrane proteins, causing a change in the active membrane transport and membrane capacity [311] leading to substantial changes in potassium, calcium, and sodium ion gradients [312], membrane potential [313], [314], cellular function [315], [316], and causing thermal block of electrically excitable cells [317], [303]. The thermo tolerable cells have significantly higher (~15%) membrane potential than

In chemo-thermo-therapies the role of chaperone proteins is important. Chaperones (stressor heat-shock-proteins) are highly conserved proteins, which are vital in almost every living cell and on their surfaces during their whole lifetime, regardless of their stage in the evolution, [318]. Any kind of change in the dynamic equilibrium of the cell life (environmental stresses, various pathogen processes, diseases, etc.) activates their synthesis [319]. Excretion of the chaperones is the 'stress-answer' of the cells to accommodate themselves to the new challenges. As a consequence of the stressful 'life' of malignant cells, the molecular chaperones are present in all cancerous cells [320], [321], [322] to adapt the actual stress to help tumor-cell survival. Moreover, the shock-proteins are induced by every oncological treatment-method, which are devoted to eliminate the malignancy: after conventional hyperthermia [323], after chemotherapy [324], after radiotherapy [325] or even after photo-therapy [326] intensive HSP synthesis was shown. By the stress adaptation the induction or over-expression of the stress proteins generally provide effective protection of the cell against apoptosis [327], but their extracellular expression acts oppositely: it makes a signal to the immune system on the defect of the actual cell [328]. Furthermore, induction of various HSPs (HSP27, HSP70, and HSP90) was observed in numerous metastases and the HSP90 homologue, GRP94 may act as a mediator of metastasis generation. HSP generally degrades the effect of the hyperthermia therapy because it may increase the tumor cell survival, and its massive induction may generate the tumor thermo-tolerance and in parallel drug- and radio-

the naïve cells [314**]**, and the difference rapidly grows by the elapsed time at 45°C.

proteins [310]. All of these events can arrest the cell-cycle of the tumor.

tolerance. Heat treatment can also lead to multi-drug resistance [329].

for the treatment response [331].

retinoblastoma protein [333].

Non-temperature dependent effects (mainly field stresses) can also produce chaperonesynthesis [330]. The HSP manifestation in the biopsies might give a good clinical indication

On the other hand, the chaperone HSP70 assists to freeze the actual dynamic equilibrium (the "status-quo") and so tries to re-establish the cellular communication in the extra-cellular electrolyte [328]. It is shown that their expression on the cell-membrane gains the apoptotic signals and enhances the immune reactions, [328]. HSP participates in the activation of the p53 tumor-suppresser [332] and has been associated with the tumor-suppresser

Recently, numerous scientific theories have also concentrated on the significance of thermally induced non-thermal effects, such as heat-shock protein (HSP) production [334], The above described mechanism targets the membrane of the malignant cells, and excites numerous pathways essential for the cellular fate, (see Figure 31*.).* The excitations have *s*erious consequences on membrane:


The effects in the cytoplasm are also remarkable. The transmembrane temperature gradient


These effects were shown in multiple, different experiments. The basic repeated phenomena are the time-delay of the action, selection and tumor destruction of oncothermia became trivial hours after the treatment (see Figure 32., [337]). The numerous apoptotic bodies show the character of the process.

Local Hyperthermia in Oncology – To Choose or not to Choose? 41

The time delay indicates the long-duration processes, which were identified as programmed cell-death (apoptosis), by various investigations: macro- and micro-morphology, enhanced activity of p53 tumor-suppressor, cleaved caspase 3 involvement, Tunel reaction, DNA fragmentation (laddering), etc. (see Figure 33., [337]). The rebuilt external apoptotic pathways are shown by reestablished E-cadherin connections, by enreachment of betacatenin and its relocalization into the nuclei, by connexin induction, by activation of death-

**Figure 33.** External electromagnetic field activates signal transduction pathways, concluding to

This apoptotic process is non-toxic (no inflammatory reactions afterwards) and promotes

The cellular selection is proven by various experiments. One of these is the definite selection of malignant cells in co-cultures, [338]. The effect on healthy human keratinocytes (HCK) cocultured with human fibroblasts (see Figure 34.) was marginal, and was regenerated by the time. The co-culture with immortalized (but not malignant) HaCaT keratinocytes is affected slightly, but the malignant A431 cell-line was selected from the co-culture (see Figure 35.). The time-delay and the relocalization of the beat-catenin to the cell-nuclei is shown in vitro

The time delay is shown in the Tunel reactions (see Figure 37.), indicating the late apoptotic

An interesting measurement was provided by Ki67 proliferation marker, measured in the living tumor part after the single-shot treatment of oncothermia, (see Figure 37, [340]). The surviving, living malignant cells in the treated tumor had definitely and significantly

apoptotic cell-death. (Oncothermia treatment (HT29 colorectal xenograft model)

(HepG2 cell-line) and in vivo (HT29 xenograft) shown in Figure 36. [339].

phases dominantly after 24 h of the single shot treatment.

the immune reactions (works parallel with these).

toll receptors, like FADD, FAS, DR-5, etc.

**Figure 31.** Numerous pathways are excited

**Figure 32.** Morphological observations show extended cell-destruction in the large volume of the tumor (field effect is active, temperature doesn't change, self-focusing). The destruction has a timedelay, hours are requested for the natural processes to be well- activated to fight. The numbers indicated on the mice are the hours after the single shot of 30 min oncothermia treatment, (HE Staining). (a) full cross section of the tumor, (b) magnification of the tissue by 100x

The time delay indicates the long-duration processes, which were identified as programmed cell-death (apoptosis), by various investigations: macro- and micro-morphology, enhanced activity of p53 tumor-suppressor, cleaved caspase 3 involvement, Tunel reaction, DNA fragmentation (laddering), etc. (see Figure 33., [337]). The rebuilt external apoptotic pathways are shown by reestablished E-cadherin connections, by enreachment of betacatenin and its relocalization into the nuclei, by connexin induction, by activation of deathtoll receptors, like FADD, FAS, DR-5, etc.

40 Hyperthermia

**Figure 31.** Numerous pathways are excited

**Figure 32.** Morphological observations show extended cell-destruction in the large volume of the tumor (field effect is active, temperature doesn't change, self-focusing). The destruction has a timedelay, hours are requested for the natural processes to be well- activated to fight. The numbers

(a) full cross section of the tumor, (b) magnification of the tissue by 100x

indicated on the mice are the hours after the single shot of 30 min oncothermia treatment, (HE Staining).

**Figure 33.** External electromagnetic field activates signal transduction pathways, concluding to apoptotic cell-death. (Oncothermia treatment (HT29 colorectal xenograft model)

This apoptotic process is non-toxic (no inflammatory reactions afterwards) and promotes the immune reactions (works parallel with these).

The cellular selection is proven by various experiments. One of these is the definite selection of malignant cells in co-cultures, [338]. The effect on healthy human keratinocytes (HCK) cocultured with human fibroblasts (see Figure 34.) was marginal, and was regenerated by the time. The co-culture with immortalized (but not malignant) HaCaT keratinocytes is affected slightly, but the malignant A431 cell-line was selected from the co-culture (see Figure 35.).

The time-delay and the relocalization of the beat-catenin to the cell-nuclei is shown in vitro (HepG2 cell-line) and in vivo (HT29 xenograft) shown in Figure 36. [339].

The time delay is shown in the Tunel reactions (see Figure 37.), indicating the late apoptotic phases dominantly after 24 h of the single shot treatment.

An interesting measurement was provided by Ki67 proliferation marker, measured in the living tumor part after the single-shot treatment of oncothermia, (see Figure 37, [340]). The surviving, living malignant cells in the treated tumor had definitely and significantly

suppressed Ki67 marker compared to its untreated counterpart in all the time-scale investigated.

Local Hyperthermia in Oncology – To Choose or not to Choose? 43

**Figure 36.** The beta-catenin relocalization into the nuclei

**Figure 37.** TUNEL assay: enzymatically label the DNA fragments resulted by apoptotic process

**Figure 38.** Colon26 (murine colorectal cancer) cell line derived allograft (CID mice), single shot. (Ki-67:

proliferation marker protein, expressed in the nuclear membrane only in the dividing cells.)

**Figure 34.** Co-culture with normal human skin fibroblasts as a model of a squamous carcinoma growing within connective tissue cells (100.000/ml) were exposed to oncothermia. Samples were incubated for 24 h at 37°C, fixed and stained with crystal violet. Cellular metabolic activity was measured using the MTT assay (standard colorimetric test) and quantitated at 630 nm. (Data represent the mean value ± S.E.M. of 4-6 separate experiments assayed in triplicate, but some experiments were repeated up to 12 times to obtain reliable data.)

**Figure 35.** The selection of the malignant cells is effective in the co-culture of the malignant A431 human keratinocytes and healthy human fibroblasts. [a – before oncothermia, b – after oncothermia]. The healthy cells remain intact, while the malignant cells are damaged and cleared. (Experiment is described in Figure 34.)

Local Hyperthermia in Oncology – To Choose or not to Choose? 43

**Figure 36.** The beta-catenin relocalization into the nuclei

42 Hyperthermia

investigated.

suppressed Ki67 marker compared to its untreated counterpart in all the time-scale

**Figure 34.** Co-culture with normal human skin fibroblasts as a model of a squamous carcinoma growing within connective tissue cells (100.000/ml) were exposed to oncothermia. Samples were incubated for 24 h at 37°C, fixed and stained with crystal violet. Cellular metabolic activity was measured using the MTT assay (standard colorimetric test) and quantitated at 630 nm. (Data represent the mean value ± S.E.M. of 4-6 separate experiments assayed in triplicate, but some experiments were

**Figure 35.** The selection of the malignant cells is effective in the co-culture of the malignant A431 human keratinocytes and healthy human fibroblasts. [a – before oncothermia, b – after oncothermia]. The healthy cells remain intact, while the malignant cells are damaged and cleared. (Experiment is

repeated up to 12 times to obtain reliable data.)

described in Figure 34.)

**Figure 37.** TUNEL assay: enzymatically label the DNA fragments resulted by apoptotic process

**Figure 38.** Colon26 (murine colorectal cancer) cell line derived allograft (CID mice), single shot. (Ki-67: proliferation marker protein, expressed in the nuclear membrane only in the dividing cells.)

The cleaved Caspase-3 (as important part of the apoptotic pathway) was activated (see Figure 39., [341]), as well as the death toll receptors became overexpressed (see Figure 40., [341]).

Local Hyperthermia in Oncology – To Choose or not to Choose? 45

**Figure 41.** Oncothermia can reestablish the adherent cell connections (E-cadherin and β-catenin) as well as the gap-junctions (Connexin-43). These could reestablish the cellular communication and can stop the

This "gluing mechanism" is not only important for the external apoptotic signal, but makes the malignant cells less mobile, their motility is reduced and the dissemination risk is

The main danger of malignancies is the metastases, attacking the organs which are crucial for life. When the tumor grows somewhere without endangering the important systems like the respiratory system, central nervous system, cardiovascular system, etc., it is not lifethreatening. These tumors are local (benign or early malignant), their elimination is possible. The real life-threatening danger is the malignancy, when the cells are disseminated from the tumor-lesion by the various transport systems (lymph, blood), or their effect becomes

The heavy life-threatening effect of metastases has been observed on statistical basis on colorectal adenocarcinoma collecting data for 15 years [342]. The long- term (10 years) survival was around 90% when no metastases were present, 60% in case of regional metastases and only 15% when distant metastases were developed in the patient. The bloodtransported cells can be blocked easily by the brain, lung, kidney, liver, etc., causing fatality. The challenge of the treatments is to recognize the tumor early, to avoid the metastases,

In veterinarian clinical trial of dogs having osteosarcomas without evidences of metastases, the local radiation combined complementary with whole body hyperthermia was studied. The result was surprisingly bad [343], [344]: the combined treatment was not effective on the

dissemination of tumor cells

**4.3. Action on far distant metastases** 

systemic by one of the general mechanisms of the organism.

and/or to block the dissemination as much as possible.

decreased.

**Figure 39.** HT29 human colorectal carcinoma cell-line xenograft model in nude mice. Caspases, or cysteine-aspartic proteases or cysteine-dependent aspartate-directed proteases are a family of cysteine proteases that play essential roles in apoptosis (programmed cell death), necrosis, and inflammation (the red line is for the reference value) [Reference: untreated control]

**Figure 40.** HT29 human colorectal carcinoma cell-line xenograft model in nude mice. Death receptor activity (the red line is for the reference value) [Reference: untreated control]
